Bispecific t cell activating antigen binding molecules

ABSTRACT

The present invention generally relates to novel bispecific antigen binding molecules for T cell activation and re-direction to specific target cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

FIELD OF THE INVENTION

The present invention generally relates to bispecific antigen bindingmolecules for activating T cells. In addition, the present inventionrelates to polynucleotides encoding such bispecific antigen bindingmolecules, and vectors and host cells comprising such polynucleotides.The invention further relates to methods for producing the bispecificantigen binding molecules of the invention, and to methods of usingthese bispecific antigen binding molecules in the treatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. CTLs constitute the most potent effector cells of the immunesystem, however they cannot be activated by the effector mechanismmediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies designed to bind with one “arm” toa surface antigen on target cells, and with the second “arm” to anactivating, invariant component of the T cell receptor (TCR) complex,have become of interest in recent years. The simultaneous binding ofsuch an antibody to both of its targets will force a temporaryinteraction between target cell and T cell, causing activation of anycytotoxic T cell and subsequent lysis of the target cell. Hence, theimmune response is re-directed to the target cells and is independent ofpeptide antigen presentation by the target cell or the specificity ofthe T cell as would be relevant for normal MHC-restricted activation ofCTLs. In this context it is crucial that CTLs are only activated when atarget cell is presenting the bispecific antibody to them, i.e. theimmunological synapse is mimicked. Particularly desirable are bispecificantibodies that do not require lymphocyte preconditioning orco-stimulation in order to elicit efficient lysis of target cells.

Several bispecific antibody formats have been developed and theirsuitability for T cell mediated immunotherapy investigated. Out ofthese, the so-called BiTE (bispecific T cell engager) molecules havebeen very well characterized and already shown some promise in theclinic (reviewed in Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260(2011)). BiTEs are tandem scFv molecules wherein two scFv molecules arefused by a flexible linker. Further bispecific formats being evaluatedfor T cell engagement include diabodies (Holliger et al., Prot Eng 9,299-305 (1996)) and derivatives thereof, such as tandem diabodies(Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recentdevelopment are the so-called DART (dual affinity retargeting)molecules, which are based on the diabody format but feature aC-terminal disulfide bridge for additional stabilization (Moore et al.,Blood 117, 4542-51 (2011)). The so-called triomabs, which are wholehybrid mouse/rat IgG molecules and also currently being evaluated inclinical trials, represent a larger sized format (reviewed in Seimetz etal., Cancer Treat Rev 36, 458-467 (2010)). The variety of formats thatare being developed shows the great potential attributed to T cellre-direction and activation in immunotherapy. The task of generatingbispecific antibodies suitable therefor is, however, by no meanstrivial, but involves a number of challenges that have to be met relatedto efficacy, toxicity, applicability and produceability of theantibodies.

Small constructs such as, for example, BiTE molecules—while being ableto efficiently crosslink effector and target cells—have a very shortserum half life requiring them to be administered to patients bycontinuous infusion. IgG-like formats on the other hand—while having thegreat benefit of a long half life—suffer from toxicity associated withthe native effector functions inherent to IgG molecules. Theirimmunogenic potential constitutes another unfavorable feature ofIgG-like bispecific antibodies, especially non-human formats, forsuccessful therapeutic development. Finally, a major challenge in thegeneral development of bispecific antibodies has been the production ofbispecific antibody constructs at a clinically sufficient quantity andpurity, due to the mispairing of antibody heavy and light chains ofdifferent specificities upon co-expression, which decreases the yield ofthe correctly assembled construct and results in a number ofnon-functional side products from which the desired bispecific antibodymay be difficult to separate. Given the difficulties and disadvantagesassociated with currently available bispecific antibodies for T cellmediated immunotherapy, there remains a need for novel, improved formatsof such molecules. The present invention provides bispecific antigenbinding molecules designed for T cell activation and re-direction thatcombine good efficacy and produceability with low toxicity and favorablepharmacokinetic properties.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to a target cell antigen.

In one embodiment the first antigen binding moiety which is a Fabmolecule capable of specific binding to CD3 comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO:33 and a variable light chain comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO:31.

In one embodiment the first antigen binding moiety which is a Fabmolecule capable of specific binding to CD3 comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 3 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7. In a specificembodiment the second antigen binding moiety is capable of specificbinding to Carcinoembryonic Antigen (CEA, CEACAM5) and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO:26 and at least one light chain CDR selected from the group of SEQ IDNO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to CEA and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 27.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP (CSPG4) and comprises at least oneheavy chain complementarity determining region (CDR) selected from thegroup consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO: 50.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to Melanoma-associated Chondroitin SulfateProteoglycan (MCSP, CSPG4) and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 18, SEQID NO: 19 and SEQ ID NO: 20.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selectedfrom the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 39 and SEQ ID NO: 41 and a light chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to an amino acid sequence selected from the group of SEQID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO:51.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 13 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 17.

In a particular embodiment, the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged. Inan even more particular embodiment, the first antigen binding moiety isa crossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In a further particular embodiment, not more than one antigen bindingmoiety capable of specific binding to CD3 is present in the T cellactivating bispecific antigen binding molecule (i.e. the T cellactivating bispecific antigen binding molecule provides monovalentbinding to CD3).

In a further embodiment said T cell activating bispecific antigenbinding molecule further comprises a third antigen binding moiety whichis a Fab molecule capable of specific binding to a target cell antigen.In one embodiment said third antigen binding molecule is a conventionalFab molecule. In one embodiment said third antigen binding molecule isidentical to the second antigen binding moiety.

In a particular embodiment said T cell activating bispecific antigenbinding molecule further comprises a third antigen binding moiety whichis a Fab molecule capable of specific binding to CEA, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO:26 and at least one light chain CDR selected from the group of SEQ IDNO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.

In a particular embodiment said T cell activating bispecific antigenbinding molecule further comprises a third antigen binding moiety whichis a Fab molecule capable of specific binding to CEA, and comprises aheavy chain variable region comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 23 and a light chain variable region comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 27.

In one embodiment said T cell activating bispecific antigen bindingmolecule further comprises a third antigen binding moiety which is a Fabmolecule capable of specific binding to MCSP, and comprises at least oneheavy chain complementarity determining region (CDR) selected from thegroup consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO: 50.

In a particular embodiment said T cell activating bispecific antigenbinding molecule further comprises a third antigen binding moiety whichis a Fab molecule capable of specific binding to MCSP, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:16 and at least one light chain CDR selected from the group of SEQ IDNO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

In one embodiment said T cell activating bispecific antigen bindingmolecule further comprises a third antigen binding moiety which is a Fabmolecule capable of specific binding to MCSP, and comprises a heavychain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence selectedfrom the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ IDNO: 47 and SEQ ID NO: 51.

In a particular embodiment said T cell activating bispecific antigenbinding molecule further comprises a third antigen binding moiety whichis a Fab molecule capable of specific binding to MCSP, and comprises aheavy chain variable region comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 13 and a light chain variable region comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the first and the second antigen binding moiety ofthe T cell activating bispecific antigen binding molecule are fused toeach other, optionally via a peptide linker. In one such embodiment, thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety. In another such embodiment, the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.In embodiments wherein either (i) the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety or (ii) the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety, additionally the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety maybe fused to each other, optionally via a peptide linker.

In one embodiment said T cell activating bispecific antigen bindingmolecule further comprises (iii) an Fc domain composed of a first and asecond subunit capable of stable association.

In one embodiment, the second antigen binding moiety of the T cellactivating bispecific antigen binding molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain. In another embodiment, the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or second subunit of the Fc domain. Inone embodiment, the first and the second antigen binding moiety of the Tcell activating bispecific antigen binding molecule are each fused atthe C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain.

In one embodiment, the third antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a particular embodiment, the secondand the third antigen binding moiety of the T cell activating antigenbinding molecule are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety. In another particular embodiment, the first and the thirdantigen binding moiety of the T cell activating antigen binding moleculeare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. The components of the T cell activating bispecific antigenbinding molecule may be fused directly or through suitable peptidelinkers. In one embodiment the first and the third antigen bindingmoiety and the Fc domain are part of an immunoglobulin molecule.

In a particular embodiment the immunoglobulin molecule is an IgG classimmunoglobulin. In an even more particular embodiment the immunoglobulinis an IgG₁ subclass immunoglobulin. In another embodiment, theimmunoglobulin is an IgG₄ subclass immunoglobulin.

In a particular embodiment, the Fc domain is an IgG Fc domain. In aspecific embodiment, the Fc domain is an IgG₁ Fc domain. In anotherspecific embodiment, the Fc domain is an IgG₄ Fc domain. In an even morespecific embodiment, the Fc domain is an IgG₄ Fc domain comprising theamino acid substitution S228P (Kabat numbering). In particularembodiments the Fc domain is a human Fc domain.

In particular embodiments the Fc domain comprises a modificationpromoting the association of the first and the second Fc domain subunit.In a specific such embodiment, an amino acid residue in the CH3 domainof the first subunit of the Fc domain is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable.

In a particular embodiment the Fc domain exhibits reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain. In certain embodiments the Fc domain isengineered to have reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a non-engineered Fc domain. Inone embodiment, the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor and/or effectorfunction. In one embodiment, the one or more amino acid substitution inthe Fc domain that reduces binding to an Fc receptor and/or effectorfunction is at one or more position selected from the group of L234,L235, and P329 (Kabat numbering). In particular embodiments, eachsubunit of the Fc domain comprises three amino acid substitutions thatreduce binding to an Fc receptor and/or effector function wherein saidamino acid substitutions are L234A, L235A and P329G. In one suchembodiment, the Fc domain is an IgG₁ Fc domain, particularly a humanIgG₁ Fc domain. In other embodiments, each subunit of the Fc domaincomprises two amino acid substitutions that reduce binding to an Fcreceptor and/or effector function wherein said amino acid substitutionsare L235E and P329G. In one such embodiment, the Fc domain is an IgG₄ Fcdomain, particularly a human IgG₄ Fc domain. In one embodiment, the Fcdomain of the T cell activating bispecific antigen binding molecule isan IgG₄ Fc domain and comprises the amino acid substitutions L235E andS228P (SPLE).

In one embodiment the Fc receptor is an Fcγ receptor. In one embodimentthe Fc receptor is a human Fc receptor. In one embodiment, the Fcreceptor is an activating Fc receptor. In a specific embodiment, the Fcreceptor is human FcγRIIa, FcγRI, and/or FcγRIIIa. In one embodiment,the effector function is antibody-dependent cell-mediated cytotoxicity(ADCC).

According to another aspect of the invention there is provided anisolated polynucleotide encoding a T cell activating bispecific antigenbinding molecule of the invention or a fragment thereof. The inventionalso encompasses polypeptides encoded by the polynucleotides of theinvention. The invention further provides an expression vectorcomprising the isolated polynucleotide of the invention, and a host cellcomprising the isolated polynucleotide or the expression vector of theinvention. In some embodiments the host cell is a eukaryotic cell,particularly a mammalian cell. In another aspect is provided a method ofproducing the T cell activating bispecific antigen binding molecule ofthe invention, comprising the steps of a) culturing the host cell of theinvention under conditions suitable for the expression of the T cellactivating bispecific antigen binding molecule and b) recovering the Tcell activating bispecific antigen binding molecule. The invention alsoencompasses a T cell activating bispecific antigen binding moleculeproduced by the method of the invention.

The invention further provides a pharmaceutical composition comprisingthe T cell activating bispecific antigen binding molecule of theinvention and a pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the T cellactivating bispecific antigen binding molecule and pharmaceuticalcomposition of the invention. In one aspect the invention provides a Tcell activating bispecific antigen binding molecule or a pharmaceuticalcomposition of the invention for use as a medicament. In one aspect isprovided a T cell activating bispecific antigen binding molecule or apharmaceutical composition according to the invention for use in thetreatment of a disease in an individual in need thereof. In a specificembodiment the disease is cancer.

Also provided is the use of a T cell activating bispecific antigenbinding molecule of the invention for the manufacture of a medicamentfor the treatment of a disease in an individual in need thereof; as wellas a method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule according to the invention in a pharmaceutically acceptableform. In a specific embodiment the disease is cancer. In any of theabove embodiments the individual preferably is a mammal, particularly ahuman.

The invention also provides a method for inducing lysis of a targetcell, particularly a tumor cell, comprising contacting a target cellwith a T cell activating bispecific antigen binding molecule of theinvention in the presence of a T cell, particularly a cytotoxic T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Exemplary configurations of the T cell activating bispecificantigen binding molecules (TCBs) of the invention. (A) Illustration ofthe “1+1 IgG Crossfab” molecule. (B) Illustration of the “2+1 IgGCrossfab” molecule. (C) Illustration of the “2+1 IgG Crossfab” moleculewith alternative order of Crossfab and Fab components (“inverted”). (D)Illustration of the “1+1 CrossMab” molecule. (E) Illustration of the“2+1 IgG Crossfab, linked light chain” molecule. (F) Illustration of the“1+1 IgG Crossfab, linked light chain” molecule. (G) Illustration of the“2+1 IgG Crossfab, inverted, linked light chain” molecule. (H)Illustration of the “1+1 IgG Crossfab, inverted, linked light chain”molecule. Black dot: optional modification in the Fc domain promotingheterodimerization.

FIG. 2. Alignment of affinity matured anti-MCSP clones compared to thenon-matured parental clone (M4-3 ML2).

FIG. 3. Schematic drawing of the MCSP TCB (2+1 Crossfab-IgG P329G LALAinverted) molecule.

FIG. 4. CE-SDS analyses of MCSP TCB (2+1 Crossfab-IgG P329G LALAinverted, SEQ ID NOs: 12, 53, 54 and 55). Electropherogram shown asSDS-Page of MCSP TCB: A) non reduced, B) reduced.

FIG. 5. Schematic drawing of CEA TCB (2+1 Crossfab-IgG P329G LALAinverted) molecule.

FIG. 6. CE-SDS analyses of CEA TCB (2+1 Crossfab-IgG P329G LALAinverted, SEQ ID NOs: 22, 56, 57 and 58) molecule. Electropherogramshown as SDS-Page of CEA TCB: A) non reduced, B) reduced.

FIG. 7. Binding of MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) to A375cells (MCSP⁺) (A) and Jurkat (CD3⁺ cells) (B). “Untargeted TCB”:bispecific antibody engaging CD3 but no second antigen (SEQ ID NOs:59,60, 61 and 62).

FIG. 8. T-cell killing induced by MCSP TCB antibody (SEQ ID NOs: 12, 53,54 and 55) of A375 (high MCSP) (A), MV-3 (medium MCSP) (B), HCT-116 (lowMCSP) (C) and LS180 (MCSP negative) (D) target cells (E:T=10:1,effectors human PBMCs, incubation time 24 h). “Untargeted TCB”:bispecific antibody engaging CD3 but no second antigen (SEQ ID NOs:59,60, 61 and 62).

FIG. 9. Upregulation of CD25 and CD69 on human CD8⁺ (A, B) and CD4⁺ (C,D) T cells after T cell-mediated killing of MV3 melanoma cells(E:T=10:1,24 h incubation) induced by MCSP TCB antibody (SEQ ID NOs: 12,53, 54 and 55). “Untargeted TCB”: bispecific antibody engaging CD3 butno second antigen (SEQ ID NOs: 59, 60, 61 and 62).

FIG. 10. Secretion of IL-2 (A), IFN-γ (B), TNFα (C), IL-4 (D), IL-10 (E)and Granzyme B (F) by human PBMCs after T cell mediated killing of MV3melanoma cells (E:T=10:1, 24 h incubation) induced by MCSP TCB antibody(SEQ ID NOs: 12, 53, 54 and 55). “Untargeted TCB”: bispecific antibodyengaging CD3 but no second antigen (SEQ ID NOs: 59, 60, 61 and 62).

FIG. 11. Binding of CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) toCEA-expressing A549 lung adenocarcinoma cells (A) and CD3-expressingimmortalized human and cynomolgus T lymphocyte lines (Jurkat (B) andHSC-F (C), respectively).

FIG. 12. T-cell killing induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and58) of HPAFII (high CEA) (A, E), BxPC-3 (medium CEA) (B, F), ASPC-1 (lowCEA) (C, G) and HCT-116 cells (CEA negative) (D, H). E:T=10:1, effectorshuman PBMCs, incubation time 24 h (A-D) or 48 h (E-H). “Untargeted TCB”:bispecific antibody engaging CD3 but no second antigen (SEQ ID NOs: 59,60, 61 and 62).

FIG. 13. Human CD8⁺ and CD4⁺ T cell proliferation (A-D) and upregulationof CD25 on human CD8⁺ and CD4 T cells (E-H) 5 days after T cell-mediatedkilling of HPAFII (high CEA) (A, E), BxPC-3 (medium CEA) (B, F), ASPC-1(low CEA) (C, G) and HCT-116 cells (CEA negative) (D, H) induced by CEATCB (SEQ ID NOs: 22, 56, 57 and 58). “DP47 TCB”: bispecific antibodyengaging CD3 but no second antigen (SEQ ID NOs: 59, 60, 61 and 62).

FIG. 14. Secretion of IFN-γ (A), TNFα (B), Granzyme B (C), IL-2 (D),IL-6 (E) and IL-10 (F) after T cell mediated killing of MKN45 tumorcells (E:T=10:1, 48 h incubation) induced by CEA TCB (SEQ ID NOs: 22,56, 57 and 58). “Untargeted TCB”: bispecific antibody engaging CD3 butno second antigen (SEQ ID NOs: 59, 60, 61 and 62).

FIG. 15. T cell-mediated killing of CEA-expressing LS180 tumor targetcells induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) in presence ofincreasing concentrations of shed CEA (sCEA), detected 24 h (A) or 48 h(B) after incubation with the CEA TCB and sCEA.

FIG. 16. T cell-mediated killing of A549 (lung adenocarcinoma) cellsoverexpressing human CEA (A549-hCEA), assessed 21 h (A, B) and 40 h (C,D) after incubation with CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) andhuman PBMCs (A, C) or cynomolgus PBMCs (B, D) as effector cells.

FIG. 17. T cell-mediated killing of CEA-expressing human colorectalcancer cell lines induced by CEA TCB (SEQ ID NOs: 22, 56, 57 and 58) at0.8 nM (A), 4 nM (B) and 20 nM (C). (D) correlation between CEAexpression and % specific lysis at 20 nM of CEA TCB, (E) correlationbetween CEA expression and EC₅₀ of CEA TCB.

FIG. 18. In vivo anti-tumor efficacy of CEA TCB (SEQ ID NOs: 22, 56, 57and 58) in a LS174T-fluc2 human colon carcinoma co-grafted with humanPBMC (E:T ratio 5:1). Results show average and SEM from 12 mice of tumorvolume measured by caliper (A and C) and by bioluminescence (Total Flux,B and D) in the different study groups. (A, B) early treatment startingat day 1, (C, D) delayed treatment starting at day 7. The MCSP TCB (SEQID NOs: 12, 53, 54 and 55) was used as negative control.

FIG. 19. In vivo anti-tumor efficacy of CEA TCB (SEQ ID NOs: 22, 56, 57and 58) in a LS174T-fluc2 human colon carcinoma co-grafted with humanPBMC (E:T ratio 1:1). Results show average and SEM from 10 mice of tumorvolume measured by caliper (A) and by bioluminescence (Total Flux, B) inthe different study groups. The MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55)was used as negative control.

FIG. 20. In vivo efficacy of murinized CEA TCB in a Panco2-huCEAorthotopic tumor model in immunocompetent huCD3c/huCEA transgenic mice.

FIG. 21. Thermal stability of CEA TCB. Dynamic Light Scattering measuredin a temperature ramp from 25-75° C. at 0.05° C./min. Duplicate is shownin grey.

FIG. 22. Thermal stability of MCSP TCB. Dynamic Light Scatteringmeasured in a temperature ramp from 25-75° C. at 0.05° C./min. Duplicateis shown as grey line.

FIG. 23. T cell-mediated killing induced by MCSP TCB (SEQ ID NOs: 12,53, 54 and 55) and MCSP 1+1 CrossMab TCB antibodies of (A) A375 (highMCSP), (B) MV-3 (medium MCSP) and (C) HCT-116 (low MCSP) tumor targetcells. (D) LS180 (MCSP negative tumor cell line) was used as negativecontrol. Tumor cell killing was assessed 24 h (A-D) and 48 h (E-H) postincubation of target cells with the antibodies and effector cells (humanPBMCs).

FIG. 24. CD25 and CD69 upregulation on CD8⁺ and CD4⁺ T cells afterT-cell killing of MCSP-expressing tumor cells (A375, A-D and MV-3, E-H)mediated by the MCSP TCB (SEQ ID NOs: 12, 53, 54 and 55) and MCSP 1+1CrossMab TCB antibodies.

FIG. 25. CE-SDS analyses of DP47 GS TCB (2+1 Crossfab-IgG P329G LALAinverted=“Untargeted TCB” SEQ ID NOs: 59, 60, 61 and 62) containing DP47GS as non binding antibody and humanized CH2527 as anti CD3 antibody.Electropherogram shown as SDS-PAGE of DP47 GS TCB: A) non reduced, B)reduced.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellor tumor stroma bearing the antigenic determinant. In another embodimentan antigen binding moiety is able to activate signaling through itstarget antigen, for example a T cell receptor complex antigen. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Particular antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may comprise antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orμ. Useful light chain constant regions include any of the two isotypes:κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein(e.g. MCSP, CEA, CD3) can be any native form the proteins from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g. mice and rats), unless otherwise indicated. In aparticular embodiment the antigen is a human protein. Where reference ismade to a specific protein herein, the term encompasses the“full-length”, unprocessed protein as well as any form of the proteinthat results from processing in the cell. The term also encompassesnaturally occurring variants of the protein, e.g. splice variants orallelic variants. Exemplary human proteins useful as antigens include,but are not limited to: Melanoma-associated Chondroitin SulfateProteoglycan (MCSP), also known as Chondroitin Sulfate Proteoglycan 4(CSPG4, UniProt no. Q6UVK1 (version 70), NCBI RefSeq no. NP_001888.2);Carcinoembroynic antigen (CEA), also known as Carcinoembryonicantigen-related cell adhesion molecule 5 (CEACAM5, UniProt no. P06731(version 119), NCBI RefSeq no. NP_004354.2); and CD3, particularly theepsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBIRefSeq no. NP_000724.1, SEQ ID NO: 103 for the human sequence; orUniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO:104 for the cynomolgus [Macaca fascicularis] sequence). In certainembodiments the T cell activating bispecific antigen binding molecule ofthe invention binds to an epitope of CD3 or a target cell antigen thatis conserved among the CD3 or target antigen from different species. Incertain embodiments the T cell activating bispecific antigen bindingmolecule of the invention binds to CD3 and CEACAM5, but does not bind toCEACAM1 or CEACAM6. By “specific binding” is meant that the binding isselective for the antigen and can be discriminated from unwanted ornon-specific interactions. The ability of an antigen binding moiety tobind to a specific antigenic determinant can be measured either throughan enzyme-linked immunosorbent assay (ELISA) or other techniquesfamiliar to one of skill in the art, e.g. surface plasmon resonance(SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al.,Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley,Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of bindingof an antigen binding moiety to an unrelated protein is less than about10% of the binding of the antigen binding moiety to the antigen asmeasured, e.g., by SPR. In certain embodiments, an antigen bindingmoiety that binds to the antigen, or an antigen binding moleculecomprising that antigen binding moiety, has a dissociation constant(K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules of the invention are capable of inducing T cell activation.Suitable assays to measure T cell activation are known in the artdescribed herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma.

As used herein, the terms “first” and “second” with respect to antigenbinding moieties etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the T cellactivating bispecific antigen binding molecule unless explicitly sostated.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In certainembodiments, one of the antigen binding moieties is a single-chain Fabmolecule, i.e. a Fab molecule wherein the Fab light chain and the Fabheavy chain are connected by a peptide linker to form a single peptidechain. In a particular such embodiment, the C-terminus of the Fab lightchain is connected to the N-terminus of the Fab heavy chain in thesingle-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein either the variable regions or the constant regions ofthe Fab heavy and light chain are exchanged, i.e. the crossover Fabmolecule comprises a peptide chain composed of the light chain variableregion and the heavy chain constant region, and a peptide chain composedof the heavy chain variable region and the light chain constant region.For clarity, in a crossover Fab molecule wherein the variable regions ofthe Fab light chain and the Fab heavy chain are exchanged, the peptidechain comprising the heavy chain constant region is referred to hereinas the “heavy chain” of the crossover Fab molecule. Conversely, in acrossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged, the peptide chaincomprising the heavy chain variable region is referred to herein as the“heavy chain” of the crossover Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant regions (VH-CH1), and a lightchain composed of the light chain variable and constant regions (VL-CL).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B 1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable A as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

The polypeptide sequences of the sequence listing are not numberedaccording to the Kabat numbering system. However, it is well within theordinary skill of one in the art to convert the numbering of thesequences of the Sequence Listing to Kabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G329, P329G, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof. The terms “host cell”, “host cell line,” and “hostcell culture” are used interchangeably and refer to cells into whichexogenous nucleic acid has been introduced, including the progeny ofsuch cells. Host cells include “transformants” and “transformed cells,”which include the primary transformed cell and progeny derived therefromwithout regard to the number of passages. Progeny may not be completelyidentical in nucleic acid content to a parent cell, but may containmutations. Mutant progeny that have the same function or biologicalactivity as screened or selected for in the originally transformed cellare included herein. A host cell is any type of cellular system that canbe used to generate the bispecific antigen binding molecules of thepresent invention. Host cells include cultured cells, e.g. mammaliancultured cells, such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YOmyeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, yeast cells, insect cells, and plant cells, to nameonly a few, but also cells comprised within a transgenic animal,transgenic plant or cultured plant or animal tissue.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, T cellactivating bispecific antigen binding molecules of the invention areused to delay development of a disease or to slow the progression of adisease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In a first aspect the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to a target cell antigen.

In one embodiment the first antigen binding moiety comprises a heavychain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQID NO: 33 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group of: SEQ IDNO: 7 and SEQ ID NO: 31.

In one embodiment the first antigen binding moiety comprises a heavychain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 3 and a light chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7.

In a specific embodiment the second antigen binding moiety is capable ofspecific binding to CEA and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and atleast one light chain CDR selected from the group of SEQ ID NO: 28, SEQID NO: 29 and SEQ ID NO: 30.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to CEA and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 27.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO: 50.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 18, SEQID NO: 19 and SEQ ID NO: 20.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selectedfrom the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 39 and SEQ ID NO: 41 and a light chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to an amino acid sequence selected from the group of SEQID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO:51.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to MCSP and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 13 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 17.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to CEA comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and atleast one light chain CDR selected from the group of SEQ ID NO: 28, SEQID NO: 29 and SEQ ID NO: 30.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7,(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to CEA comprising heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 27.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to MCSP comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 18, SEQID NO: 19 and SEQ ID NO: 20.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to MCSP comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 17.

In a particular embodiment, the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In a particular embodiment, the first antigen binding moiety is acrossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged, and the second antigenbinding moiety is a conventional Fab molecule. In a further particularembodiment, the first and the second antigen binding moiety are fused toeach other, optionally through a peptide linker.

In particular embodiments, the T cell activating bispecific antigenbinding molecule further comprises an Fc domain composed of a first anda second subunit capable of stable association.

In a further particular embodiment, not more than one antigen bindingmoiety capable of specific binding to CD3 is present in the T cellactivating bispecific antigen binding molecule (i.e. the T cellactivating bispecific antigen binding molecule provides monovalentbinding to CD3).

T Cell Activating Bispecific Antigen Binding Molecule Formats

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1, 3 and 5.

In particular embodiments, the T cell activating bispecific antigenbinding molecule comprises an Fc domain composed of a first and a secondsubunit capable of stable association. In some embodiments, the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or the second subunit of the Fc domain.

In one such embodiment, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding moiety. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or the second subunit of the Fc domain.Optionally, the Fab light chain of the first antigen binding moiety andthe Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

In another such embodiment, the first antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof a first and a second antigen binding moiety, an Fc domain composed ofa first and a second subunit, and optionally one or more peptidelinkers, wherein the first and the second antigen binding moiety areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofone of the subunits of the Fc domain.

In other embodiments, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain.

In a particular such embodiment, the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or the second subunit of the Fc domain.Optionally, the Fab light chain of the first antigen binding moiety andthe Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally a number between 1 and 10,typically between 2 and 4. A particularly suitable peptide linker forfusing the Fab light chains of the first and the second antigen bindingmoiety to each other is (G₄S)₂. An exemplary peptide linker suitable forconnecting the Fab heavy chains of the first and the second antigenbinding moiety is EPKSC(D)-(G₄S)₂ (SEQ ID NOs 105 and 106).Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where an antigen binding moiety is fused tothe N-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

A T cell activating bispecific antigen binding molecule with a singleantigen binding moiety capable of specific binding to a target cellantigen (for example as shown in FIG. 1A, 1D, 1F or 1H) is useful,particularly in cases where internalization of the target cell antigenis to be expected following binding of a high affinity antigen bindingmoiety. In such cases, the presence of more than one antigen bindingmoiety specific for the target cell antigen may enhance internalizationof the target cell antigen, thereby reducing its availability.

In many other cases, however, it will be advantageous to have a T cellactivating bispecific antigen binding molecule comprising two or moreantigen binding moieties specific for a target cell antigen (seeexamples in shown in FIG. 1B, 1C, 1E or 1G), for example to optimizetargeting to the target site or to allow crosslinking of target cellantigens.

Accordingly, in certain embodiments, the T cell activating bispecificantigen binding molecule of the invention further comprises a thirdantigen binding moiety which is a Fab molecule capable of specificbinding to a target cell antigen. In one embodiment, the third antigenbinding moiety is a conventional Fab molecule. In one embodiment, thethird antigen binding moiety is capable of specific binding to the sametarget cell antigen as the second antigen binding moiety. In aparticular embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to a target cell antigen. In aparticular embodiment, the second and the third antigen binding moietyare identical (i.e. they comprise the same amino acid sequences).

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to CEA, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and atleast one light chain CDR selected from the group of SEQ ID NO: 28, SEQID NO: 29 and SEQ ID NO: 30.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10; and the second and third antigen binding moieties arecapable of specific binding to CEA, wherein the second and third antigenbinding moieties comprise at least one heavy chain complementaritydetermining region (CDR) selected from the group consisting of SEQ IDNO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDRselected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:30.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10; and the second and third antigen binding moieties arecapable of specific binding to CEA, wherein the second and third antigenbinding moieties comprise at least one heavy chain complementaritydetermining region (CDR) selected from the group consisting of SEQ IDNO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and at least one light chain CDRselected from the group of SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:30.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to an amino acid sequence selected from thegroup of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31, andthe second and third antigen binding moieties are capable of specificbinding to CEA, wherein the second and third antigen binding moietiescomprise a heavy chain variable region comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 23 and a light chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3,and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7, and the second and third antigen bindingmoieties are capable of specific binding to CEA, wherein the second andthird antigen binding moieties comprise a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 27.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to MCSP, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO: 50. In a particular embodiment, the first antigenbinding moiety is capable of specific binding to CD3, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6and at least one light chain CDR selected from the group of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10; and the second and third antigen bindingmoieties are capable of specific binding to MCSP, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO: 50. In one embodiment, the first antigen bindingmoiety is capable of specific binding to CD3, and comprises at least oneheavy chain complementarity determining region (CDR) selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and atleast one light chain CDR selected from the group of SEQ ID NO: 8, SEQID NO: 9, SEQ ID NO: 10; and the second and third antigen bindingmoieties are capable of specific binding to MCSP, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 18, SEQID NO: 19 and SEQ ID NO: 20.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to an amino acid sequence selected from thegroup of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO: 31, andthe second and third antigen binding moieties are capable of specificbinding to MCSP, wherein the second and third antigen binding moietiescomprise a heavy chain variable region comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to anamino acid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46,SEQ ID NO: 47 and SEQ ID NO: 51.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7, and the second and third antigen bindingmoieties are capable of specific binding to MCSP, wherein the second andthird antigen binding moieties comprise a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 17.

In one embodiment, the third antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a more specific embodiment, thesecond and the third antigen binding moiety are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain, and the first antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the second antigen binding moiety. Optionally, the Fablight chain of the first antigen binding moiety and the Fab light chainof the second antigen binding moiety may additionally be fused to eachother.

The second and the third antigen binding moiety may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe second and the third antigen binding moiety are each fused to the Fcdomain through an immunoglobulin hinge region. In a specific embodiment,the immunoglobulin hinge region is a human IgG₁ hinge region. In oneembodiment the second and the third antigen binding moiety and the Fcdomain are part of an immunoglobulin molecule. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In another embodiment the immunoglobulin is anIgG₄ subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin. In one embodiment, the T cell activating bispecificantigen binding molecule essentially consists of an immunoglobulinmolecule capable of specific binding to a target cell antigen, and anantigen binding moiety capable of specific binding to CD3 wherein theantigen binding moiety is a Fab molecule, particularly a crossover Fabmolecule, fused to the N-terminus of one of the immunoglobulin heavychains, optionally via a peptide linker.

In a particular embodiment, the first and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. In a specific such embodiment, the T cell activating bispecificantigen binding molecule essentially consists of a first, a second and athird antigen binding moiety, an Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety, and the first antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe second subunit of the Fc domain. Optionally, the Fab light chain ofthe first antigen binding moiety and the Fab light chain of the secondantigen binding moiety may additionally be fused to each other.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and the lightchain CDR 3 of SEQ ID NO: 10, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to CEA comprising the heavy chainCDR 1 of SEQ ID NO: 24, the heavy chain CDR 2 of SEQ ID NO: 25, theheavy chain CDR 3 of SEQ ID NO: 26, the light chain CDR 1 of SEQ ID NO:28, the light chain CDR 2 of SEQ ID NO: 29 and the light chain CDR3 ofSEQ ID NO: 30.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to CEA comprising heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 23 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 27.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chain CDR 1of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and the lightchain CDR 3 of SEQ ID NO: 10, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to MCSP comprising the heavy chainCDR 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, theheavy chain CDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO:18, the light chain CDR 2 of SEQ ID NO: 19 and the light chain CDR3 ofSEQ ID NO: 20.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to MCSP comprising a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 13 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 17.

The T cell activating bispecific antigen binding molecule according toany of the four above embodiments may further comprise (iii) an Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, and the first antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.

In some of the T cell activating bispecific antigen binding molecule ofthe invention, the Fab light chain of the first antigen binding moietyand the Fab light chain of the second antigen binding moiety are fusedto each other, optionally via a linker peptide. Depending on theconfiguration of the first and the second antigen binding moiety, theFab light chain of the first antigen binding moiety may be fused at itsC-terminus to the N-terminus of the Fab light chain of the secondantigen binding moiety, or the Fab light chain of the second antigenbinding moiety may be fused at its C-terminus to the N-terminus of theFab light chain of the first antigen binding moiety. Fusion of the Fablight chains of the first and the second antigen binding moiety furtherreduces mispairing of unmatched Fab heavy and light chains, and alsoreduces the number of plasmids needed for expression of some of the Tcell activating bispecific antigen binding molecules of the invention.

In certain embodiments the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab light chain variableregion of the first antigen binding moiety shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the firstantigen binding moiety (i.e. a the first antigen binding moietycomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region), which in turnshares a carboxy-terminal peptide bond with an Fc domain subunit(VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)), and a polypeptide wherein a the Fab heavychain of the second antigen binding moiety shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab heavy chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (VH₍₁₎-CL₍₁₎) and the Fab lightchain polypeptide of the second antigen binding moiety (VL₍₂₎-CL₍₂₎). Incertain embodiments the polypeptides are covalently linked, e.g., by adisulfide bond.

In alternative embodiments the T cell activating bispecific antigenbinding molecule comprises a polypeptide wherein the Fab heavy chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (i.e. the first antigen bindingmoiety comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the second antigen binding moiety shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab light chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first antigen binding moiety (VL₍₁₎-CH1₍₁₎) and the Fab lightchain polypeptide of the second antigen binding moiety (VL₍₂₎-CL₍₂₎). Incertain embodiments the polypeptides are covalently linked, e.g., by adisulfide bond.

In some embodiments, the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab light chain variableregion of the first antigen binding moiety shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the firstantigen binding moiety (i.e. the first antigen binding moiety comprisesa crossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the secondantigen binding moiety, which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit(VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In other embodiments, the Tcell activating bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain variable region of the firstantigen binding moiety shares a carboxy-terminal peptide bond with theFab light chain constant region of the first antigen binding moiety(i.e. the first antigen binding moiety comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the second antigen binding moiety,which in turn shares a carboxy-terminal peptide bond with an Fc domainsubunit (VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In still otherembodiments, the T cell activating bispecific antigen binding moleculecomprises a polypeptide wherein the Fab heavy chain of the secondantigen binding moiety shares a carboxy-terminal peptide bond with theFab light chain variable region of the first antigen binding moietywhich in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the first antigen binding moiety (i.e. thefirst antigen binding moiety comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). Inother embodiments, the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab heavy chain of thesecond antigen binding moiety shares a carboxy-terminal peptide bondwith the Fab heavy chain variable region of the first antigen bindingmoiety which in turn shares a carboxy-terminal peptide bond with the Fablight chain constant region of the first antigen binding moiety (i.e.the first antigen binding moiety comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)).

In some of these embodiments the T cell activating bispecific antigenbinding molecule further comprises a crossover Fab light chainpolypeptide of the first antigen binding moiety, wherein the Fab heavychain variable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (VH₍₁₎-CL₍₁₎), and the Fab lightchain polypeptide of the second antigen binding moiety (VL₍₂₎-CL₍₂₎). Inothers of these embodiments the T cell activating bispecific antigenbinding molecule further comprises a crossover Fab light chainpolypeptide, wherein the Fab light chain variable region of the firstantigen binding moiety shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the first antigen binding moiety(VL₍₁₎-CH1₍₁₎), and the Fab light chain polypeptide of the secondantigen binding moiety (VL₍₂₎-CL₍₂₎). In still others of theseembodiments the T cell activating bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab light chain variableregion of the first antigen binding moiety shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the firstantigen binding moiety which in turn shares a carboxy-terminal peptidebond with the Fab light chain polypeptide of the second antigen bindingmoiety (VL₍₁₎-CH1₍₁₎-VL₍₂₎-CL₍₂₎), a polypeptide wherein the Fab heavychain variable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe second antigen binding moiety (VH₍₁₎-CL₍₁₎-VL₍₂₎-CL₍₂₎), apolypeptide wherein the Fab light chain polypeptide of the secondantigen binding moiety shares a carboxy-terminal peptide bond with theFab light chain variable region of the first antigen binding moietywhich in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the first antigen binding moiety(VL₍₂₎-CL₍₂₎-VL₍₁₎-CH1₍₁₎), or a polypeptide wherein the Fab light chainpolypeptide of the second antigen binding moiety shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the first antigen binding moiety which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (VL₍₂₎-CL₍₂₎-VH₍₁₎-CL₍₁₎).

The T cell activating bispecific antigen binding molecule according tothese embodiments may further comprise (i) an Fc domain subunitpolypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavychain of a third antigen binding moiety shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) andthe Fab light chain polypeptide of a third antigen binding moiety(VL₍₃₎-CL₍₃₎). In certain embodiments the polypeptides are covalentlylinked, e.g., by a disulfide bond.

According to any of the above embodiments, components of the T cellactivating bispecific antigen binding molecule (e.g. antigen bindingmoiety, Fc domain) may be fused directly or through various linkers,particularly peptide linkers comprising one or more amino acids,typically about 2-20 amino acids, that are described herein or are knownin the art. Suitable, non-immunogenic peptide linkers include, forexample, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n) peptide linkers,wherein n is generally a number between 1 and 10, typically between 2and 4.

Fc Domain

The Fc domain of the T cell activating bispecific antigen bindingmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the T cell activating bispecific antigenbinding molecule of the invention comprises not more than one Fc domain.In one embodiment according the invention the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG Fc domain. In aparticular embodiment the Fc domain is an IgG₁ Fc domain. In anotherembodiment the Fc domain is an IgG₄ Fc domain. In a more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position 5228 (Kabat numbering), particularly the aminoacid substitution S228P. This amino acid substitution reduces in vivoFab arm exchange of IgG₄ antibodies (see Stubenrauch et al., DrugMetabolism and Disposition 38, 84-91 (2010)). In a further particularembodiment the Fc domain is human. An exemplary sequence of a human IgG₁Fc region is given in SEQ ID NO: 107.

Fc Domain Modifications Promoting Heterodimerization

T cell activating bispecific antigen binding molecules according to theinvention comprise different antigen binding moieties, fused to one orthe other of the two subunits of the Fc domain, thus the two subunits ofthe Fc domain are typically comprised in two non-identical polypeptidechains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of T cell activatingbispecific antigen binding molecules in recombinant production, it willthus be advantageous to introduce in the Fc domain of the T cellactivating bispecific antigen binding molecule a modification promotingthe association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecule according to theinvention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the T cell activating bispecific antigenbinding molecule an amino acid residue is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). In one embodiment, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter, J Immunol Methods248, 7-15 (2001)).

In a particular embodiment the antigen binding moiety capable of bindingto CD3 is fused (optionally via the antigen binding moiety capable ofbinding to a target cell antigen) to the first subunit of the Fc domain(comprising the “knob” modification). Without wishing to be bound bytheory, fusion of the antigen binding moiety capable of binding to CD3to the knob-containing subunit of the Fc domain will (further) minimizethe generation of antigen binding molecules comprising two antigenbinding moieties capable of binding to CD3 (steric clash of twoknob-containing polypeptides).

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the T cell activating bispecific antigenbinding molecule favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the T cell activatingbispecific antigen binding molecule to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties andthe long half-life of the antigen binding molecule, results in excessiveactivation of cytokine receptors and severe side effects upon systemicadministration. Activation of (Fc receptor-bearing) immune cells otherthan T cells may even reduce efficacy of the T cell activatingbispecific antigen binding molecule due to the potential destruction ofT cells e.g. by NK cells.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecules according to theinvention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG₁ Fc domain domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fey receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or the T cellactivating bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the T cell activating bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the T cell activating bispecific antigen binding moleculecomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one embodiment the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In one embodiment the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of the Fc domainto the Fc receptor, the combination of these amino acid mutations mayreduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the T cell activating bispecific antigen binding moleculecomprising an engineered Fc domain exhibits less than 20%, particularlyless than 10%, more particularly less than 5% of the binding affinity toan Fc receptor as compared to a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain. In a particularembodiment the Fc receptor is an Fcγ receptor. In some embodiments theFc receptor is a human Fc receptor. In some embodiments the Fc receptoris an activating Fc receptor. In a specific embodiment the Fc receptoris an activating human Fey receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the Fc domain to saidreceptor, is achieved when the Fc domain (or the T cell activatingbispecific antigen binding molecule comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the T cell activating bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or T cell activating bispecific antigen binding moleculesof the invention comprising said Fc domain, may exhibit greater thanabout 80% and even greater than about 90% of such affinity. In certainembodiments the Fc domain of the T cell activating bispecific antigenbinding molecule is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329. In a more specific embodiment the Fcdomain comprises an amino acid substitution at a position selected fromthe group of L234, L235 and P329. In some embodiments the Fc domaincomprises the amino acid substitutions L234A and L235A. In one suchembodiment, the Fc domain is an IgG₁ Fc domain, particularly a humanIgG₁ Fc domain. In one embodiment the Fc domain comprises an amino acidsubstitution at position P329. In a more specific embodiment the aminoacid substitution is P329A or P329G, particularly P329G. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329 and a further amino acid substitution at a positionselected from E233, L234, L235, N297 and P331. In a more specificembodiment the further amino acid substitution is E233P, L234A, L235A,L235E, N297A, N297D or P331S. In particular embodiments the Fc domaincomprises amino acid substitutions at positions P329, L234 and L235. Inmore particular embodiments the Fc domain comprises the amino acidmutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment,the Fc domain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain.The “P329G LALA” combination of amino acid substitutions almostcompletely abolishes Fcγ receptor (as well as complement) binding of ahuman IgG₁ Fc domain, as described in PCT publication no. WO2012/130831, incorporated herein by reference in its entirety. WO2012/130831 also describes methods of preparing such mutant Fc domainsand methods for determining its properties such as Fc receptor bindingor effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position 5228, specificallythe amino acid substitution S228P. To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E. Inanother embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position P329, specifically the amino acid substitutionP329G. In a particular embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at positions S228, L235 and P329, specifically aminoacid substitutions S228P, L235E and P329G. Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G.

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or a T cell activating bispecificantigen binding molecule comprising an Fc domain, can be measured bymethods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PB MC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the T cell activating bispecificantigen binding molecule is able to bind C1q and hence has CDC activity.See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Antigen Binding Moieties

The antigen binding molecule of the invention is bispecific, i.e. itcomprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants. According to theinvention, the antigen binding moieties are Fab molecules (i.e. antigenbinding domains composed of a heavy and a light chain, each comprising avariable and a constant region). In one embodiment said Fab moleculesare human. In another embodiment said Fab molecules are humanized. Inyet another embodiment said Fab molecules comprise human heavy and lightchain constant regions.

At least one of the antigen binding moieties is a crossover Fabmolecule. Such modification prevent mispairing of heavy and light chainsfrom different Fab molecules, thereby improving the yield and purity ofthe T cell activating bispecific antigen binding molecule of theinvention in recombinant production. In a particular crossover Fabmolecule useful for the T cell activating bispecific antigen bindingmolecule of the invention, the constant regions of the Fab light chainand the Fab heavy chain are exchanged. In another crossover Fab moleculeuseful for the T cell activating bispecific antigen binding molecule ofthe invention, the variable regions of the Fab light chain and the Fabheavy chain are exchanged.

In a particular embodiment according to the invention, the T cellactivating bispecific antigen binding molecule is capable ofsimultaneous binding to a target cell antigen, particularly a tumor cellantigen, and CD3. In one embodiment, the T cell activating bispecificantigen binding molecule is capable of crosslinking a T cell and atarget cell by simultaneous binding to a target cell antigen and CD3. Inan even more particular embodiment, such simultaneous binding results inlysis of the target cell, particularly a tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the T cell activatingbispecific antigen binding molecule to CD3 without simultaneous bindingto the target cell antigen does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen bindingmolecule is capable of re-directing cytotoxic activity of a T cell to atarget cell. In a particular embodiment, said re-direction isindependent of MHC-mediated peptide antigen presentation by the targetcell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

CD3 Binding Moiety

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to CD3 (also referred to herein as an “CD3 antigen bindingmoiety” or “first antigen binding moiety”). In a particular embodiment,the T cell activating bispecific antigen binding molecule comprises notmore than one antigen binding moiety capable of specific binding to CD3.In one embodiment the T cell activating bispecific antigen bindingmolecule provides monovalent binding to CD3. The CD3 antigen binding isa crossover Fab molecule, i.e. a Fab molecule wherein either thevariable or the constant regions of the Fab heavy and light chains areexchanged. In embodiments where there is more than one antigen bindingmoiety capable of specific binding to a target cell antigen comprised inthe T cell activating bispecific antigen binding molecule, the antigenbinding moiety capable of specific binding to CD3 preferably is acrossover Fab molecule and the antigen binding moieties capable ofspecific binding to a target cell antigen are conventional Fabmolecules.

In a particular embodiment CD3 is human CD3 (SEQ ID NO: 103) orcynomolgus CD3 (SEQ ID NO: 104), most particularly human CD3. In aparticular embodiment the CD3 antigen binding moiety is cross-reactivefor (i.e. specifically binds to) human and cynomolgus CD3. In someembodiments, the first antigen binding moiety is capable of specificbinding to the epsilon subunit of CD3.

The CD3 antigen binding moiety comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10.

In one embodiment the CD3 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 of SEQ ID NO: 5, theheavy chain CDR3 of SEQ ID NO: 6, the light chain CDR1 of SEQ ID NO: 8,the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ IDNO: 10.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to an amino acid sequence selected from the group of:SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33, and a light chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to an amino acid sequence selected from the group of:SEQ ID NO: 7 and SEQ ID NO: 31.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a lightchain variable region comprising an amino acid sequence selected fromthe group of: SEQ ID NO: 7 and SEQ ID NO: 31.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 3 and a light chain variable regionsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 7.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 3 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 7.

In one embodiment the CD3 antigen binding moiety comprises the heavychain variable region sequence of SEQ ID NO: 3 and the light chainvariable region sequence of SEQ ID NO: 7.

Target Cell Antigen Binding Moiety

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to a target cell antigen (also referred to herein as an “targetcell antigen binding moiety” or “second” or “third” antigen bindingmoiety). In certain embodiments, the T cell activating bispecificantigen binding molecule comprises two antigen binding moieties capableof binding to a target cell antigen. In a particular such embodiment,each of these antigen binding moieties specifically binds to the sameantigenic determinant. In an even more particular embodiment, all ofthese antigen binding moieties are identical. In one embodiment, the Tcell activating bispecific antigen binding molecule comprises animmunoglobulin molecule capable of specific binding to a target cellantigen. In one embodiment the T cell activating bispecific antigenbinding molecule comprises not more than two antigen binding moietiescapable of binding to a target cell antigen.

The target cell antigen binding moiety is generally a Fab molecule,particularly a conventional Fab molecule that binds to a specificantigenic determinant and is able to direct the T cell activatingbispecific antigen binding molecule to a target site, for example to aspecific type of tumor cell that bears the antigenic determinant.

In certain embodiments the target cell antigen binding moietyspecifically binds to a cell surface antigen. In a particular embodimentthe target cell antigen binding moiety specifically binds to amembrane-proximal region of a cell surface antigen. In a specific suchembodiment the cell surface antigen is Carcinoembryonic Antigen (CEA)and the membrane-proximal region is the B3 domain of CEA (residues208-286 of SEQ ID NO: 119). In another specific such embodiment the cellsurface antigen is Melanoma-associated Chondroitin Sulfate Proteoglycan(MCSP) and the membrane-proximal region is the D3 domain of MSCP (SEQ IDNO: 118).

In certain embodiments the target cell antigen binding moiety isdirected to an antigen associated with a pathological condition, such asan antigen presented on a tumor cell or on a virus-infected cell.Suitable antigens are cell surface antigens, for example, but notlimited to, cell surface receptors. In particular embodiments theantigen is a human antigen. In a specific embodiment the target cellantigen is selected from Melanoma-associated Chondroitin SulfateProteoglycan (MCSP, CSPG4) and Carcinoembryonic Antigen (CEA, CEACAM5).

In some embodiments the T cell activating bispecific antigen bindingmolecule comprises at least one antigen binding moiety that is specificfor Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). In oneembodiment, the antigen binding moiety that is specific for MCSPcomprises at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQID NO: 40 and at least one light chain CDR selected from the group ofSEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.

In one embodiment, the antigen binding moiety that is specific for MCSPcomprises at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15and SEQ ID NO: 16 and at least one light chain CDR selected from thegroup of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

In one embodiment, the antigen binding moiety that is specific for MCSPcomprises the heavy chain CDR1 of SEQ ID NO: 14, the heavy chain CDR2 ofSEQ ID NO: 15, the heavy chain CDR3 of SEQ ID NO: 16, the light chainCDR1 of SEQ ID NO: 18, the light chain CDR2 of SEQ ID NO: 19, and thelight chain CDR3 of SEQ ID NO: 20.

In a further embodiment, the antigen binding moiety that is specific forMCSP comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variableregion sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group of SEQ IDNO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 51.

In a further embodiment, the antigen binding moiety that is specific forMCSP comprises a heavy chain variable region comprising an amino acidsequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chain variableregion comprising an amino acid sequence selected from the group of SEQID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO:51.

In a further embodiment, the antigen binding moiety that is specific forMCSP comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:13 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 17 or variants thereofthat retain functionality.

In one embodiment, the antigen binding moiety that is specific for MCSPcomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 13 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 17.

In one embodiment, the antigen binding moiety that is specific for MCSPcomprises the heavy chain variable region sequence of SEQ ID NO: 13 andthe light chain variable region sequence of SEQ ID NO: 17.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 53, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 55.

In particular embodiments the T cell activating bispecific antigenbinding molecule comprises at least one antigen binding moiety that isspecific for Carcinoembryonic Antigen (CEA). In one embodiment, theantigen binding moiety that is specific for CEA comprises at least oneheavy chain complementarity determining region (CDR) selected from thegroup consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 andat least one light chain CDR selected from the group of SEQ ID NO: 28,SEQ ID NO: 29 and SEQ ID NO: 30.

In one embodiment, the antigen binding moiety that is specific for CEAcomprises the heavy chain CDR1 of SEQ ID NO: 24, the heavy chain CDR2 ofSEQ ID NO: 25, the heavy chain CDR3 of SEQ ID NO: 26, the light chainCDR1 of SEQ ID NO: 28, the light chain CDR2 of SEQ ID NO: 29, and thelight chain CDR3 of SEQ ID NO: 30.

In a further embodiment, the antigen binding moiety that is specific forCEA comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 27, or variants thereofthat retain functionality.

In one embodiment, the antigen binding moiety that is specific for CEAcomprises a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 23 and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 27.

In one embodiment, the antigen binding moiety that is specific for CEAcomprises the heavy chain variable region sequence of SEQ ID NO: 23 andthe light chain variable region sequence of SEQ ID NO: 27.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 56, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 58.

Polynucleotides

The invention further provides isolated polynucleotides encoding a Tcell activating bispecific antigen binding molecule as described hereinor a fragment thereof. In some embodiments, said fragment is an antigenbinding fragment.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97 and 98 including functional fragments orvariants thereof.

The polynucleotides encoding T cell activating bispecific antigenbinding molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire T cell activating bispecificantigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional T cell activatingbispecific antigen binding molecule. For example, the light chainportion of an antigen binding moiety may be encoded by a separatepolynucleotide from the portion of the T cell activating bispecificantigen binding molecule comprising the heavy chain portion of theantigen binding moiety, an Fc domain subunit and optionally (part of)another antigen binding moiety. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe antigen binding moiety. In another example, the portion of the Tcell activating bispecific antigen binding molecule comprising one ofthe two Fc domain subunits and optionally (part of) one or more antigenbinding moieties could be encoded by a separate polynucleotide from theportion of the T cell activating bispecific antigen binding moleculecomprising the other of the two Fc domain subunits and optionally (partof) an antigen binding moiety. When co-expressed, the Fc domain subunitswill associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entire Tcell activating bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptides comprised in the T cell activatingbispecific antigen binding molecule according to the invention asdescribed herein.

In another embodiment, the present invention is directed to an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NOs 3, 7, 13, 17, 23, 27, 31, 32, 33, 34,36, 39, 41, 43, 46, 47 or 51 In another embodiment, the presentinvention is directed to an isolated polynucleotide encoding a T cellactivating bispecific antigen binding molecule or fragment thereof,wherein the polynucleotide comprises a sequence that encodes apolypeptide sequence as shown in SEQ ID NOs 22, 56, 57, 58, 12, 53, 54and 55 In another embodiment, the invention is further directed to anisolated polynucleotide encoding a T cell activating bispecific antigenbinding molecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequenceshown in SEQ ID NOs 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97 or 98. In another embodiment, the invention is directedto an isolated polynucleotide encoding a T cell activating bispecificantigen binding molecule of the invention or a fragment thereof, whereinthe polynucleotide comprises the nucleic acid sequence shown in SEQ IDNOs 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97or 98. In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence in SEQ ID NOs 3, 7, 13, 17, 23,27, 31, 32, 33, 34, 36, 39, 41, 43, 46, 47 or 51. In another embodiment,the invention is directed to an isolated polynucleotide encoding a Tcell activating bispecific antigen binding molecule or a fragmentthereof, wherein the polynucleotide comprises a sequence that encodes apolypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% identical to the amino acid sequence in SEQ ID NOs 22, 56, 57,58, 12, 53, 54 or 55. The invention encompasses an isolatedpolynucleotide encoding a T cell activating bispecific antigen bindingmolecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes the variable regionsequence of SEQ ID NOs SEQ ID NOs 3, 7, 13, 17, 23, 27, 31, 32, 33, 34,36, 39, 41, 43, 46, 47 or 51 with conservative amino acid substitutions.The invention also encompasses an isolated polynucleotide encoding a Tcell activating bispecific antigen binding molecule of the invention ora fragment thereof, wherein the polynucleotide comprises a sequence thatencodes the polypeptide sequence of SEQ ID NOs 22, 56, 57, 58, 12, 53,54 or 55 with conservative amino acid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

T cell activating bispecific antigen binding molecules of the inventionmay be obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of a T cellactivating bispecific antigen binding molecule (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe T cell activating bispecific antigen binding molecule (fragment)(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment) of theinvention, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit a-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the T cell activating bispecific antigen binding molecule is desired,DNA encoding a signal sequence may be placed upstream of the nucleicacid encoding a T cell activating bispecific antigen binding molecule ofthe invention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase. Exemplary amino acid and polynucleotide sequences ofsecretory signal peptides are given in SEQ ID NOs 108-116.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the Tcell activating bispecific antigen binding molecule may be includedwithin or at the ends of the T cell activating bispecific antigenbinding molecule (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a T cell activatingbispecific antigen binding molecule of the invention. As used herein,the term “host cell” refers to any kind of cellular system which can beengineered to generate the T cell activating bispecific antigen bindingmolecules of the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of T cell activatingbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the T cell activating bispecific antigenbinding molecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO, NS0,Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing a T cell activating bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the T cell activating bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of the T cellactivating bispecific antigen binding molecule, and recovering the Tcell activating bispecific antigen binding molecule from the host cell(or host cell culture medium).

The components of the T cell activating bispecific antigen bindingmolecule are genetically fused to each other. T cell activatingbispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of T cell activating bispecific antigen binding molecules arefound in the sequences provided herein. Additional sequences may also beincluded to incorporate a cleavage site to separate the individualcomponents of the fusion if desired, for example an endopeptidaserecognition sequence.

In certain embodiments the one or more antigen binding moieties of the Tcell activating bispecific antigen binding molecules comprise at leastan antibody variable region capable of binding an antigenic determinant.Variable regions can form part of and be derived from naturally ornon-naturally occurring antibodies and fragments thereof. Methods toproduce polyclonal antibodies and monoclonal antibodies are well knownin the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”,Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodiescan be constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the T cell activatingbispecific antigen binding molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the T cell activating bispecific antigen bindingmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g. an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. CD3) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. V9 antibody, described in U.S.Pat. No. 6,054,297) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

T cell activating bispecific antigen binding molecules prepared asdescribed herein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusion chromatography,and the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity etc., and will be apparent to those having skill in theart. For affinity chromatography purification an antibody, ligand,receptor or antigen can be used to which the T cell activatingbispecific antigen binding molecule binds. For example, for affinitychromatography purification of T cell activating bispecific antigenbinding molecules of the invention, a matrix with protein A or protein Gmay be used. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate a T cell activatingbispecific antigen binding molecule essentially as described in theExamples. The purity of the T cell activating bispecific antigen bindingmolecule can be determined by any of a variety of well known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the heavy chain fusionproteins expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing SDS-PAGE (see e.g.FIG. 4). Three bands were resolved at approximately Mr 25,000, Mr 50,000and Mr 75,000, corresponding to the predicted molecular weights of the Tcell activating bispecific antigen binding molecule light chain, heavychain and heavy chain/light chain fusion protein.

Assays

T cell activating bispecific antigen binding molecules provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the T cell activating bispecific antigen bindingmolecule for an Fc receptor or a target antigen can be determined inaccordance with the methods set forth in the Examples by surface plasmonresonance (SPR), using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. Alternatively, binding of T cellactivating bispecific antigen binding molecules for different receptorsor target antigens may be evaluated using cell lines expressing theparticular receptor or target antigen, for example by flow cytometry(FACS). A specific illustrative and exemplary embodiment for measuringbinding affinity is described in the following and in the Examplesbelow.

According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (Qiagen) immobilized on CM5 chips and the bispecific constructsare used as analytes. Briefly, carboxymethylated dextran biosensor chips(CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to40 μg/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the bispecificconstruct (range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CM5-sensor chip surface as described forthe anti Penta-His antibody. The final amount of coupled protein isapproximately 12000 RU. The bispecific constructs are captured for 90 sat 300 nM. The target antigens are passed through the flow cells for 180s at a concentration range from 250 to 1000 nM with a flowrate of 30μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the T cell activating bispecific antigen bindingmolecules of the invention can be measured by various assays asdescribed in the Examples. Biological activities may for example includethe induction of proliferation of T cells, the induction of signaling inT cells, the induction of expression of activation markers in T cells,the induction of cytokine secretion by T cells, the induction of lysisof target cells such as tumor cells, and the induction of tumorregression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the T cell activating bispecific antigen bindingmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises anyof the T cell activating bispecific antigen binding molecules providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical composition comprises any of the T cell activatingbispecific antigen binding molecules provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Further provided is a method of producing a T cell activating bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining a T cellactivating bispecific antigen binding molecule according to theinvention, and (b) formulating the T cell activating bispecific antigenbinding molecule with at least one pharmaceutically acceptable carrier,whereby a preparation of T cell activating bispecific antigen bindingmolecule is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more T cell activatingbispecific antigen binding molecule dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one T cell activating bispecificantigen binding molecule and optionally an additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standardsor corresponding authorities in other countries. Preferred compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. T cell activating bispecific antigen binding molecules of thepresent invention (and any additional therapeutic agent) can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrasplenically, intrarenally, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g. aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g. liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the T cell activatingbispecific antigen binding molecules of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the T cell activating bispecific antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the T cell activatingbispecific antigen binding molecules may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Sterile injectable solutions are prepared by incorporatingthe T cell activating bispecific antigen binding molecules of theinvention in the required amount in the appropriate solvent with variousof the other ingredients enumerated below, as required. Sterility may bereadily accomplished, e.g., by filtration through sterile filtrationmembranes. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the T cellactivating bispecific antigen binding molecules may also be formulatedas a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, the Tcell activating bispecific antigen binding molecules may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The T cell activating bispecific antigen binding molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the T cell activating bispecific antigen binding moleculesprovided herein may be used in therapeutic methods. T cell activatingbispecific antigen binding molecules of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, T cell activating bispecific antigenbinding molecules of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, T cell activating bispecific antigen binding molecules ofthe invention for use as a medicament are provided. In further aspects,T cell activating bispecific antigen binding molecules of the inventionfor use in treating a disease are provided. In certain embodiments, Tcell activating bispecific antigen binding molecules of the inventionfor use in a method of treatment are provided. In one embodiment, theinvention provides a T cell activating bispecific antigen bindingmolecule as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides a T cell activating bispecific antigen binding molecule for usein a method of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe T cell activating bispecific antigen binding molecule. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. In further embodiments, the invention provides a T cellactivating bispecific antigen binding molecule as described herein foruse in inducing lysis of a target cell, particularly a tumor cell. Incertain embodiments, the invention provides a T cell activatingbispecific antigen binding molecule for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of the Tcell activating bispecific antigen binding molecule to induce lysis of atarget cell. An “individual” according to any of the above embodimentsis a mammal, preferably a human.

In a further aspect, the invention provides for the use of a T cellactivating bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention. Inone embodiment a composition is administered to said individual,comprising the T cell activating bispecific antigen binding molecule ofthe invention in a pharmaceutically acceptable form. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments may bea mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with a T cell activatingbispecific antigen binding molecule of the invention in the presence ofa T cell, particularly a cytotoxic T cell. In a further aspect, a methodfor inducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule to induce lysis of atarget cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a T cellactivating bispecific antigen binding molecule of the present inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer. Askilled artisan readily recognizes that in many cases the T cellactivating bispecific antigen binding molecule may not provide a curebut may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount of Tcell activating bispecific antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of a T cell activatingbispecific antigen binding molecule of the invention is administered toa cell. In other embodiments, a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of aT cell activating bispecific antigen binding molecule of the invention(when used alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofT cell activating bispecific antigen binding molecule, the severity andcourse of the disease, whether the T cell activating bispecific antigenbinding molecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the T cell activating bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The T cell activating bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the T cell activating bispecific antigen bindingmolecule would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other non-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the T cell activating bispecific antigen binding molecule). Aninitial higher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The T cell activating bispecific antigen binding molecules of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, the Tcell activating bispecific antigen binding molecules of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the T cell activating bispecific antigen bindingmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the T cell activating bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecificantigen binding molecules described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a T cell activating bispecific antigen bindingmolecule can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. T cellactivating bispecific antigen binding molecules that exhibit largetherapeutic indices are preferred. In one embodiment, the T cellactivating bispecific antigen binding molecule according to the presentinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with T cell activatingbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The T cell activating bispecific antigen binding molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a T cell activating bispecific antigenbinding molecule of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent. Such other agents are suitably present incombination in amounts that are effective for the purpose intended. Theeffective amount of such other agents depends on the amount of T cellactivating bispecific antigen binding molecule used, the type ofdisorder or treatment, and other factors discussed above. The T cellactivating bispecific antigen binding molecules are generally used inthe same dosages and with administration routes as described herein, orabout from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the T cell activating bispecific antigen bindingmolecule of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. T cell activating bispecific antigen binding molecules of theinvention can also be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a T cell activating bispecific antigen binding moleculeof the invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises a Tcell activating bispecific antigen binding molecule of the invention;and (b) a second container with a composition contained therein, whereinthe composition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.Exemplary leader peptides and polynucleotide sequences encoding them aredepicted SEQ ID NOs 108-116.

Isolation of Primary Human Pan T Cells from PBMCs

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. Briefly, blood was diluted with sterile PBS and carefullylayered over a Histopaque gradient (Sigma, H8889). After centrifugationfor 30 minutes at 450×g at room temperature (brake switched off), partof the plasma above the PBMC containing interphase was discarded. ThePBMCs were transferred into new 50 ml Falcon tubes and tubes were filledup with PBS to a total volume of 50 ml. The mixture was centrifuged atroom temperature for 10 minutes at 400×g (brake switched on). Thesupernatant was discarded and the PBMC pellet washed twice with sterilePBS (centrifugation steps at 4° C. for 10 minutes at 350×g). Theresulting PBMC population was counted automatically (ViCell) and storedin RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine(Biochrom, K0302) at 37° C., 5% CO₂ in the incubator until assay start.

T cell enrichment from PBMCs was performed using the Pan T CellIsolation Kit II (Miltenyi Biotec #130-091-156), according to themanufacturer's instructions. Briefly, the cell pellets were diluted in40 μl cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA,sterile filtered) and incubated with 10 μl Biotin-Antibody Cocktail per10 million cells for 10 min at 4° C. 30 μl cold buffer and 20 μlAnti-Biotin magnetic beads per 10 million cells were added, and themixture incubated for another 15 min at 4° C. Cells were washed byadding 10-20× the current volume of buffer and a subsequentcentrifugation step at 300×g for 10 min. Up to 100 million cells wereresuspended in 500 μl buffer. Magnetic separation of unlabeled human panT cells was performed using LS columns (Miltenyi Biotec #130-042-401)according to the manufacturer's instructions. The resulting T cellpopulation was counted automatically (ViCell) and stored in AIM-V mediumat 37° C., 5% CO₂ in the incubator until assay start (not longer than 24h).

Isolation of Primary Human Naive T Cells from PBMCs

Peripheral blood mononuclar cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. T-cell enrichment from PBMCs was performed using the NaiveCD8⁺ T cell isolation Kit from Miltenyi Biotec (#130-093-244), accordingto the manufacturer's instructions, but skipping the last isolation stepof CD8⁺ T cells (also see description for the isolation of primary humanpan T cells).

Isolation of Murine Pan T Cells from Splenocytes

Spleens were isolated from C57BL/6 mice, transferred into a GentleMACSC-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5%BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtainsingle-cell suspensions according to the manufacturer's instructions.The cell suspension was passed through a pre-separation filter to removeremaining undissociated tissue particles. After centrifugation at 400×gfor 4 min at 4° C., ACK Lysis Buffer was added to lyse red blood cells(incubation for 5 min at room temperature). The remaining cells werewashed with MACS buffer twice, counted and used for the isolation ofmurine pan T cells. The negative (magnetic) selection was performedusing the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861),following the manufacturer's instructions. The resulting T cellpopulation was automatically counted (ViCell) and immediately used forfurther assays.

Isolation of Primary Cynomolgus PBMCs from Heparinized Blood

Peripheral blood mononuclar cells (PBMCs) were prepared by densitycentrifugation from fresh blood from healthy cynomolgus donors, asfollows: Heparinized blood was diluted 1:3 with sterile PBS, andLymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterilePBS. Two volumes of the diluted blood were layered over one volume ofthe diluted density gradient and the PBMC fraction was separated bycentrifugation for 30 min at 520×g, without brake, at room temperature.The PBMC band was transferred into a fresh 50 ml Falcon tube and washedwith sterile PBS by centrifugation for 10 min at 400×g at 4° C. Onelow-speed centrifugation was performed to remove the platelets (15 minat 150×g, 4° C.), and the resulting PBMC population was automaticallycounted (ViCell) and immediately used for further assays.

Target Cells

For the assessment of MCSP-targeting bispecific antigen bindingmolecules, the following tumor cell lines were used: the human melanomacell line WM266-4 (ATCC #CRL-1676), derived from a metastatic site of amalignant melanoma and expressing high levels of human MCSP; the humanmelanoma cell line MV-3 (a kind gift from The Radboud UniversityNijmegen Medical Centre), expressing medium levels of human MCSP; thehuman malignant melanoma (primary tumour) cell line A375 (ECACC#88113005) expressing high levels of MCSP; the human colon carcinomacell line HCT-116 (ATCC #CCL-247) that does not express MCSP; and thehuman Caucasian colon adenocarcinoma cell line LS180 (ECACC #87021202)that does not express MCSP. For the assessment of CEA-targetingbispecific antigen binding molecules, the following tumor cell lineswere used: the human gastric cancer cell line MKN45 (DSMZ #ACC 409),expressing very high levels of human CEA; the human pancreasadenocarcinoma cell line HPAF-II (kind gift of Roche Nutley), expressinghigh levels of human CEA; the human primary pancreatic adenocarcinomacell line BxPC-3 (ECACC #93120816) expressing medium levels of humanCEA; the human female Caucasian colon adenocarcinoma cell line LS-174T(ECACC #87060401), expressing medium levels of human CEA; the humanpancreas adenocarcinoma cell line ASPC-1 (ECACC #96020930) expressinglow levels of human CEA; the human epithelioid pancreatic carcinoma cellline Panc-1 (ATCC #CRL-1469), expressing (very) low levels of human CEA;the human colon carcinoma cell line HCT-116 (ATCC #CCL-247) that doesnot express CEA; a human adenocarcinomic alveolar basal epithelial cellline A549-huCEA that was stably transfected in-house to express humanCEA; and a murine colon carcinoma cell line MC38-huCEA, that wasengineered in-house to stably express human CEA.

In addition, a human T cell leukaemia cell line, Jurkat (ATCC #TIB-152),was used to assess binding of different bispecific constructs to humanCD3 on cells.

Example 1 Affinity Maturation of Anti-MCSP Antibody M4-3/ML2

Affinity maturation was performed via the oligonucleotide-directedmutagenesis procedure. For this the heavy chain variant M4-3, and thelight chain variant ML2 were cloned into a phagemid vector, similar tothose described by Hoogenboom, (Hoogenboom et al., Nucleic Acids Res.1991, 19, 4133-4137). Residues to be randomized were identified by firstgenerating a 3D model of that antibody via classical homology modelingand then identifying the solvent accessible residues of thecomplementary determining regions (CDRs) of heavy and light chain.Oligonucleotides with randomization based on trinucleotide synthesis asshown in Table 1 were purchased from Ella Biotech (Munich, Germany).Three independent sublibraries were generated via classical PCR, andcomprised randomization in CDR-H1 together with CDR-H2, or CDR-L1together with CDR-L2. CDR-L3 was randomized in a separate approach. TheDNA fragments of those libraries were cloned into the phagemid viarestriction digest and ligation, and subsequently electroporated intoTG1 bacteria.

Library Selection

The antibody variants thus generated were displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants were then screened for their biological activities (here:binding affinity) and candidates that have one or more improvedactivities were used for further development. Methods for making phagedisplay libraries can be found in Lee et al., J. Mol. Biol. (2004) 340,1073-1093. Selections with all affinity maturation libraries werecarried out in solution according to the following procedure: 1. bindingof ˜1012 phagemid particles of each affinity maturation libraries to 100nM biotinylated hu-MCSP(D3 domain)-avi-his (SEQ ID NO: 118) for 0.5 h ina total volume of 1 ml, 2. capture of biotinylated hu-MCSP(D3domain)-avi-his and specifically bound phage particles by addition of5.4×10⁷ streptavidin-coated magnetic beads for 10 min, 3. washing ofbeads using 5-10×1 ml PBS/Tween-20 and 5-10×1 ml PBS, 4. elution ofphage particles by addition of 1 ml 100 mM TEA (triethylamine) for 10min and neutralization by adding 500 μl 1M Tris/HCl pH 7.4 and 5.re-infection of exponentially growing E. coli TG1 bacteria, infectionwith helper phage VCSM13 and subsequent PEG/NaCl precipitation ofphagemid particles to be used in subsequent selection rounds. Selectionswere carried out over 3-5 rounds using either constant or decreasing(from 10⁻⁷ M to 2×10⁻⁹ M) antigen concentrations. In round 2, capture ofantigen-phage complexes was performed using neutravidin plates insteadof streptavidin beads. Specific binders were identified by ELISA asfollows: 100 μl of 10 nM biotinylated hu-MCSP(D3 domain)-avi-his perwell were coated on neutravidin plates. Fab-containing bacterialsupernatants were added and binding Fabs were detected via theirFlag-tags by using an anti-Flag/HRP secondary antibody. ELISA-positiveclones were bacterially expressed as soluble Fab fragments in 96-wellformat and supernatants were subjected to a kinetic screening experimentby SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing Fabswith the highest affinity constants were identified and thecorresponding phagemids were sequenced.

TABLE 1 (excluded were always Cys and Met. Lys was excluded on top inthose cases where the oligonucleotide was a reverse primer) PositionRandomization Heavy chain CDR1 Ser31 S (40%), rest (60%, 4% each) Gly32G (40%), rest (60%, 4% each). Tyr33 Y (40%), rest (60%, 4% each) Tyr34 Y(40%), rest (60%, 4% each) CDR2 Tyr50 Y 40%, (F, W, L, A, I, 30%, 6%each), rest (30%, 2.5% each) Thr52 T (60%), rest (40%, 2.5% each) Tyr53Y (40%), rest (60%, 3.8% each) Asp54 D (40%), rest (60%, 3.8% each)Ser56 S (40%), rest (60%, 3.8% each) Light chain CDR1 Gln27 Q (40%), (E,D, N, S, T, R, 40%, 6.7% each), rest (total 20%, 2.2% each) Gly28 G(40%), (N, T, S, Q, Y, D, E, 40%, 5.7% each), rest (20%, 2.5% each)Asn31 N (40%), (S, T, G, Q, Y, D, E, R, 50%, 6.3% each), rest (10%, 1.4%each) Tyr32 Y (40%), (W, S, R, 30%, 10% each), rest (30%, 2.3% each)CDR2 Tyr50 Y (70%), (E, R, K, A, Q, T, S, D, G, W, F, 30%, 2.7% each)Thr51 T (50%), (S, A, G, N, Q, V, 30%, 5% each), rest (20%, 2% each)Ser52 S (50%), rest (50%, 3.1% each) Ser53 S (40%), (N, T, Q, Y, D, E,I, 40%, 5.7% each), rest (20%, 2.2% each) CDR3 Tyr91 Y (50%), rest (50%,3.1% each) Ser92 S (50%), (N, Q, T, A, G 25%, 5% each), rest (25%, 2.3%each) Lys93 K (50%), S (5%), T (5%), N (5%), rest (35%, 2.7% each) Leu94L (50%), (Y, F, S, I, A, V, 30%, 5% each), rest (20%, 2% each) Pro95 P(50%), (S, A, 20%, 10% each), rest (30%, 2.1% each) Trp96 W 50%, (Y, R,L, 15%, 5% each), rest (35%, 2.5% each)

FIG. 2 shows an alignment of affinity matured anti-MCSP clones comparedto the non-matured parental clone (M4-3 ML2). Heavy chain randomizationwas performed only in the CDR1 and 2. Light chain randomization wasperformed in CDR1 and 2, and independently in CDR3.

During selection, a few mutations in the frameworks occured like F71Y inclone G3 or Y87H in clone E10.

Production and Purification of Human IgG₁

The variable region of heavy and light chain DNA sequences of theaffinity matured variants were subcloned in frame with either theconstant heavy chain or the constant light chain pre-inserted into therespective recipient mammalian expression vector. The antibodyexpression was driven by an MPSV promoter and carries a synthetic polyAsignal sequence at the 3′ end of the CDS. In addition each vectorcontained an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with themammalian expression vectors using polyethylenimine (PEI). The cellswere transfected with the corresponding expression vectors in a 1:1ratio. For transfection HEK293 EBNA cells were cultivated in suspensionserum-free in CD CHO culture medium. For the production in 500 ml shakeflask, 400 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection cells were centrifuged for 5 min at210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution, the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 500 ml shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. After incubation time 160 mlF17 medium was added and cells were cultivated for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was added.After 7 days cultivation supernatant was collected for purification bycentrifugation for 15 min at 210×g, the solution was sterile filtered(0.22 μm filter) and sodium azide in a final concentration of 0.01% w/vwas added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A. Supernatant was loaded on aHiTrap Protein A HP column (CV=5 mL, GE Healthcare) equilibrated with 40ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,pH 7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, pH 7.5. Target protein was eluted during a gradient over 20column volumes from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solutionwas neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Targetprotein was concentrated and filtrated prior loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of molecules were analyzed by CE-SDS analyses inthe presence and absence of a reducing agent. The Caliper LabChip GXIIsystem (Caliper Life Sciences) was used according to the manufacturer'sinstruction. 2 μg sample was used for analyses. The aggregate content ofantibody samples was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) in 25 mM K₂HPO₄, 125 mM NaCl, 200 mML-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at25° C.

TABLE 2 Production and purification of affinity matured anti-MCSP IgGsYield HMW LMW Monomer Construct [mg/l] [%] [%] [%] M4-3(C1) ML2(G3) 43.90 0 100 M4-3(C1) ML2(E10) 59.5 0 0 100 M4-3(C1) ML2(C5) 68.9 0 0.8 99.2

Affinity Determination ProteOn Analysis

K_(D) was measured by surface plasmon resonance using a ProteOn XPR36machine (BioRad) at 25° C. with anti-human F(ab′)2 fragment specificcapture antibody (Jackson ImmunoResearch #109-005-006) immobilized byamine coupling on CM5 chips and subsequent capture of Fabs frombacterial supernatant or from purified Fab preparations. Briefly,carboxymethylated dextran biosensor chips (CM5, GE Healthcare) wereactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-human F(ab′)2 fragment specific captureantibody was diluted with 10 mM sodium acetate, pH 5.0 at 50 μg/mlbefore injection at a flow rate of 10 μl/minute to achieve approximatelyup to 10.000 response units (RU) of coupled capture antibody. Followingthe injection of the capture antibody, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, Fabs from bacterialsupernatant or purified Fabs were injected at a flow rate of 10μl/minute for 300 s and a dissociation of 300 s for capture baselinestabilization. Capture levels were in the range of 100-500 RU. In asubsequent step, human MCSP(D3 domain)-avi-his analyte was injectedeither as a single concentration or as a concentration series (dependingof clone affinity in a range between 100 nM and 250 pM) diluted intoHBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Surfactant P20, pH 7.4) at 25° C. at a flow rate of 50 μl/min. Thesurface of the sensorchip was regenerated by injection of glycine pH 1.5for 30 s at 90 μl/min followed by injection of NaOH for 20 s at the sameflow rate. Association rates (k_(on)) and dissociation rates (k_(off))were calculated using a simple one-to-one Langmuir binding model(ProteOn XPR36 Evaluation Software or Scrubber software (BioLogic)) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). This data was used to determine the comparative bindingaffinity of the affinity matured variants with the parental antibody.Table 3a shows the data generated from these assays.

G3, E10, C5 for the light chain, and D6, A7, B7, B8, C1 for the heavychain were chosen for conversion into human IgG₁ format. Since CDR1 and2 of the light chain were randomized independent from CDR3, the obtainedCDRs were combined during IgG conversion.

In the IgG format affinities were measured again to the human MCSPantigen (SEQ ID NO: 118), in addition also to the cynomolgus homologue(SEQ ID NO: 117).

The method used was exactly as described for the Fab fragments, justusing purified IgG from mammalian production.

TABLE 3a MCSP affinity matured clones: Proteon data. Human Human CynoHuman Cyno MCSP MCSP MCSP MCSP MCSP Fab K_(D) IgG K_(D) IgG K_(D) IgGK_(D) IgG K_(D) Proteon Comparative binding generated affinity—FoldVariant affinity data increase over parent Parental M4-3/ML2 5 * 10⁻⁹2 * 10⁻⁹ 2 * 10⁻⁹ M4-3/ML2(G3) 4 * 10⁻¹⁰ 3 * 10⁻¹⁰ 6 * 10⁻¹⁰ 6.7 3.3M4-3/ML2(E10) 7 * 10⁻¹⁰ 1 * 10⁻⁹ 2 * 10⁻⁹ 2.0 1.0 M4- 4 * 10⁻¹⁰ 9 *10⁻¹⁰ 5.0 2.2 3/ML2(E10/G3) M4-3/ML2(C5) 7 * 10⁻¹⁰ 4 * 10⁻¹⁰ 1 * 10⁻⁹5.0 2.0 M4- 7 * 10⁻¹⁰ 1 * 10⁻⁹ 2.9 2.0 3/ML2(C5/G3) M4-3(D6)/ML2 2 *10⁻⁹ 4 * 10⁻¹⁰ 1 * 10⁻⁹ 5.0 2.0 M4-3(A7)/ML2 2 * 10⁻¹¹ 8 * 10⁻¹⁰ 1 *10⁻⁹ 2.5 2.0 M4-3(B7)/ML2 5 * 10⁻¹⁰ 7 * 10⁻¹⁰ 4.0 2.9 M4-3(B8)/ML2 3 *10⁻¹⁰ 9 * 10⁻¹⁰ 1 * 10⁻⁹ 2.2 2.0 M4-3(C1)/ML2 6 * 10⁻¹⁰ 9 * 10⁻¹⁰ 8 *10⁻¹⁰ 2.2 2.5 M4- 7 * 10⁻¹¹ 2 * 10⁻¹⁰ 28.6 10.0 3(C1)/ML2(G3) M4- 5 *10⁻¹⁰ 6 * 10⁻¹⁰ 4.0 3.3 3(C1)/ML2(E10) M4- 7 * 10⁻¹¹ 2 * 10⁻¹⁰ 28.6 10.03(A7)/ML2(G3) M4- 3 * 10⁻¹⁰ 7 * 10⁻¹⁰ 6.7 2.9 3(A7)/ML2(E10) M4- 2 *10⁻¹⁰ 3 * 10⁻¹⁰ 10.0 6.7 3(C1)/ML2(C5) M4- 7 * 10⁻¹¹ 2 * 10⁻¹⁰ 28.6 10.03(A7)/ML2(C5)

Affinity Determination by Surface Plasmon Resonance (SPR) Using BiacoreT200

Surface plasmon resonance (SPR) experiments to determine the affinityand the avidity of the affinity matured IgGs were performed on a BiacoreT200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

For analyzing the avidity of the interaction of different anti-MCSP IgGsto human and cynomolgus MCSP D3 direct coupling of around 9,500resonance units (RU) of the anti-Penta His antibody (Qiagen) wasperformed on a CM5 chip at pH 5.0 using the standard amine coupling kit(Biacore, Freiburg/Germany). Antigens were captured for 60 s at 30 nMwith 10 μl/min respectively. IgGs were passed at a concentration of0.0064-100 nM with a flowrate of 30 μl/min through the flow cells over280 s. The dissociation was monitored for 180 s. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell. Here, the IgGs were flown over a surface withimmobilized anti-Penta His antibody but on which HBS-EP has beeninjected rather than human MCSP D3 or cynomolgus MCSP D3.

For affinity measurements IgGs were captured on a CM5 sensorchip surfacewith immobilized anti human Fc. Capture IgG was coupled to thesensorchip surface by direct immobilization of around 9,500 resonanceunits (RU) at pH 5.0 using the standard amine coupling kit (Biacore,Freiburg/Germany). IgGs are captured for 25 s at 10 nM with 30 μl/min.Human and cynomolgus MCSP D3 were passed at a concentration of 2-500 nMwith a flowrate of 30 μl/min through the flow cells over 120 s. Thedissociation was monitored for 60 s. Association and dissociation forconcentration 166 and 500 nM was monitored for 1200 and 600 s,respectively. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantigens were flown over a surface with immobilized anti-human Fcantibody but on which HBS-EP has been injected rather than anti-MCSPIgGs.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration.

Higher affinity to human and cynomolgus MCSP D3 were confirmed bysurface plasmon resonance measurements using Biacore T200. In addition,avidity measurements showed an up to 3-fold increase in bivalent binding(Table 3b).

TABLE 3b Affinity and avidity of anti MCSP IgGs to human MCSP-D3 andcynomolgus MCSP-D3. KD in nM Human MCSP D3 Cynomolgus MCSP D3 T = 25° C.Affinity Avidity Affinity Avidity M4-3(C1) ML2(G3) 1.8 0.0045 1.4 0.0038M4-3(C1) ML2(E10) 4.6 0.0063 3.8 0.0044 M4-3(C1) ML2(C5) 1.8 0.0046 1.30.0044 M4-3 ML2 (parental) 8.6 0.0090 11.4 0.0123

Example 2 Preparation of MCSP TCB (2+1 Crossfab-IgG P329G LALA Inverted)Containing M4-3(C1) ML2(G3) as Anti MCSP Antibody and Humanized CH2527as Anti CD3 Antibody

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the constant heavy chain or the constantlight chain pre-inserted into the respective recipient mammalianexpression vector. The antibody expression was driven by an MPSVpromoter and carries a synthetic polyA signal sequence at the 3′ end ofthe CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with themammalian expression vectors using polyethylenimine (PEI). The cellswere transfected with the corresponding expression vectors in a 1:2:1:1ratio (“vector heavy chain Fc(hole)”:“vector light chain”:“vector lightchain Crossfab”: “vector heavy chain Fc(knob)-FabCrossfab”).

For transfection HEK293 EBNA cells were cultivated in suspensionserum-free in CD CHO culture medium. For the production in 500 ml shakeflask 400 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection cells were centrifuged for 5 min at210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 500 ml shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. After incubation time 160 mlF17 medium was added and cell were cultivated for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was added.After 7 days cultivation supernatant was collected for purification bycentrifugation for 15 min at 210×g, the solution was sterile filtered(0.22 μm filter) and sodium azide in a final concentration of 0.01% w/vwas added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A. Supernatant was loaded on aHiTrap Protein A HP column (CV=5 mL, GE Healthcare) equilibrated with 40ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,pH 7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, pH 7.5. Target protein was eluted during a gradient over 20column volumes from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solutionwas neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Targetprotein was concentrated and filtrated prior loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of molecules were analyzed by CE-SDSanalyses in the presence and absence of a reducing agent. The CaliperLabChip GXII system (Caliper lifescience) was used according to themanufacturer's instruction. 2 μg sample was used for analyses.

The aggregate content of antibody samples was analyzed using a TSKgelG3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C.

TABLE 4a Summary production and purification of MCSP TCB. Aggregateafter 1^(st) Titer Yield purification HMW LMW Monomer Construct [mg/l][mg/l] step [%] [%] [%] [%] MCSP TCB 157 0.32 32 3.3 0 96.7

FIG. 3 shows a schematic drawing of the MCSP TCB (2+1 Crossfab-IgG P329GLALA inverted) molecule.

FIG. 4 and Table 4b show CE-SDS analyses of a MCSP TCB (2+1 Crossfab-IgGP329G LALA inverted) molecule (SEQ ID NOs: 12, 53, 54 and 55).

TABLE 4b CE-SDS analyses of MCSP TCB. Peak kDa Corresponding Chain MCSPTCB non reduced (A) 1 206.47 MCSP TCB reduced (B) 1 29.15 Light chainML2 (C1) 2 37.39 Light chain huCH2527 3 66.07 Fc(hole) 4 94.52 Fc(knob)

Example 3 Preparation of CEA TCB (2+1 Crossfab-IgG P329G LALA Inverted)Containing CH1A1A 98/99 2F1 as Anti CEA Antibody and Humanized CH2527 asAnti CD3 Antibody

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the constant heavy chain or the constantlight chain pre-inserted into the respective recipient mammalianexpression vector. The antibody expression was driven by an MPSVpromoter and carries a synthetic polyA signal sequence at the 3′ end ofthe CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293 EBNA cells with themammalian expression vectors using polyethylenimine (PEI). The cellswere transfected with the corresponding expression vectors in a 1:2:1:1ratio (“vector heavy chain Fc(hole)”:“vector light chain”:“vector lightchain Crossfab”: “vector heavy chain Fc(knob)-FabCrossfab”).

For transfection HEK293 EBNA cells were cultivated in suspensionserum-free in CD CHO culture medium. For the production in 500 ml shakeflask 400 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection cells were centrifuged for 5 min at210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 500 ml shake flask and incubated for 3 hours by 37° C.in an incubator with a 5% CO₂ atmosphere. After incubation time 160 mlF17 medium was added and cell were cultivated for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was added.After 7 days cultivation supernatant was collected for purification bycentrifugation for 15 min at 210×g, the solution was sterile filtered(0.22 μm filter) and sodium azide in a final concentration of 0.01% w/vwas added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A. Supernatant was loaded on aHiTrap Protein A HP column (CV=5 mL, GE Healthcare) equilibrated with 40ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,pH 7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, pH 7.5. Target protein was eluted during a gradient over 20column volumes from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solutionwas neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Targetprotein was concentrated and filtrated prior loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of molecules were analyzed by CE-SDSanalyses in the presence and absence of a reducing agent. The CaliperLabChip GXII system (Caliper lifescience) was used according to themanufacturer's instructions. 2 μg sample was used for analyses.

The aggregate content of antibody samples was analyzed using a TSKgelG3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C.

TABLE 5 Summary production and purification of CEA TCB. Aggregate after1^(st) Titer Yield purification HMW LMW Monomer Construct [mg/l] [mg/l]step [%] [%] [%] [%] CEA TCB 66 0.31 21.5 8.1 4.4 87.5

FIG. 5 shows a schematic drawing of CEA TCB (2+1 Crossfab-IgG P329G LALAinverted) molecule.

FIG. 6 and Table 6 show CE-SDS analyses of a CEA TCB (2+1 Crossfab-IgGP329G LALA inverted) molecule (SEQ ID NOs: 22, 56, 57 and 58).

TABLE 6 CE-SDS analyses of CEA TCB. Peak kDa Corresponding Chain CEA TCBnon reduced (A) 1 205.67 Correct molecule CEA TCB reduced (B) 1 28.23Light chain CH1A1A 98/99 × 2F1 2 36.31 Light chain CH2527 3 63.48Fc(hole) 4 90.9 Fc(knob)

In an alternative purification method, the CEA TCB was captured fromharvested and clarified fermentation supernatant by Protein A affinitychromatography (MabSelect SuRe). The Protein A eluate was then submittedto cation exchange chromatography (Poros 50 HS) and subsequentlyfractionated and analyzed by means of SE-HPLC. and capillaryelectrophoresis. The product containing fractions were pooled andsubjected to hydrophobic interaction chromatography (Butyl-Sepharose4FF) at room temperature in a bind-elute mode. The eluate therefrom wasthen fractionated and analyzed by means of SE-HPLC and capillaryelectrophoresis. The product containing fractions were pooled andsubsequently anion exchange chromatography (Q-Sepharose FF) inflow-through mode was performed. The material obtained using thispurification method had a monomer content of >98%.

Example 4 Binding of MCSP TCB to MCSP- and CD3-Expressing Cells

The binding of MCSP TCB was tested on a MCSP-expressing human malignantmelanoma cell line (A375) and a CD3-expressing immortalized T lymphocyteline (Jurkat). Briefly, cells were harvested, counted, checked forviability and resuspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS0.1% BSA). 100 μl cell suspension (containing 0.2×10⁶ cells) wereincubated in round-bottom 96-well plate for 30 min at 4° C. withincreasing concentrations of the MCSP TCB (2.6 pM-200 nM), washed twicewith cold PBS 0.1% BSA, re-incubated for further 30 min at 4° C. withthe PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG FcγFragment Specific secondary antibody (Jackson Immuno Research Lab PE#109-116-170), washed twice with cold PBS 0.1% BSA and immediatelyanalyzed by FACS using a FACS CantoII (Software FACS Diva) by gatinglive, DAPI-negative, cells. Binding curves were obtained usingGraphPadPrism5 (FIG. 7A, binding to A375 cells, EC₅₀=3381 pM; FIG. 7B,binding to Jurkat cells).

Example 5 T-Cell Killing Induced by MCSP TCB Antibody

T-cell killing mediated by MCSP TCB antibody was assessed using a panelof tumor cell lines expressing different levels of MCSP (A375=MCSP high,MV-3=MSCP medium, HCT-116=MCSP low, LS180=MCSP negative). Briefly,target cells were harvested with Trypsin/EDTA, washed, and plated atdensity of 25 000 cells/well using flat-bottom 96-well plates. Cellswere left to adhere overnight. Peripheral blood mononuclear cells(PBMCs) were prepared by Histopaque density centrifugation of enrichedlymphocyte preparations (buffy coats) obtained from healthy humandonors. Fresh blood was diluted with sterile PBS and layered overHistopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30minutes, room temperature), the plasma above the PBMC-containinginterphase was discarded and PBMCs transferred in a new falcon tubesubsequently filled with 50 ml of PBS. The mixture was centrifuged(400×g, 10 minutes, room temperature), the supernatant discarded and thePBMC pellet washed twice with sterile PBS (centrifugation steps 350×g,10 minutes). The resulting PBMC population was counted automatically(ViCell) and stored in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO₂ in cellincubator until further use (no longer than 24 h). For the killingassay, the antibody was added at the indicated concentrations (range of1 pM-10 nM in triplicates). PBMCs were added to target cells at finaleffector to target (E:T) ratio of 10:1. Target cell killing was assessedafter 24 h of incubation at 37° C., 5% CO₂ by quantification of LDHreleased into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells with1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubatedwith effector cells without bispecific construct. The results show thatMCSP TCB induced a strong and target-specific killing of MCSP-positivetarget cell lines with no killing of MCSP-negative cell lines (FIG. 8,A-D). The EC₅₀ values related to the killing assays, calculated usingGraphPadPrism5 are given in Table 7.

TABLE 7 EC₅₀ values (pM) for T-cell mediated killing of MCSP- expressingtumor cells induced by MCSP TCB antibody. Cell line MCSP receptor copynumber EC₅₀ [pM] A375 387 058 12.3 MV-3 260 000 9.4 HCT-116 36770 3.7LS180 Negative n.d.

Example 6

CD25 and CD69 Upregulation on CD8⁺ and CD4⁺ Effector Cells after T CellKilling of MCSP-Expressing Tumor Cells Induced by MCSP TCB Antibody

Activation of CD8⁺ and CD4⁺ T cells after T-cell killing ofMCSP-expressing MV-3 tumor cells mediated by the MCSP TCB antibody wasassessed by FACS analysis using antibodies recognizing the T cellactivation markers CD25 (late activation marker) and CD69 (earlyactivation marker). The antibody and the killing assay conditions wereessentially as described above (Example 5), using the same antibodyconcentration range (1 pM-10 nM in triplicates), E:T ratio 10:1 and anincubation time 24 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 350×g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (FITC anti-human CD8, BD#555634), CD4 (PECy7 anti-human CD4, BD #557852), CD69 (PE anti-humanCD69, Biolegend #310906) and CD25 (APC anti-human CD25, BD #555434) wasperformed according to the suppliers' indications. Cells were washedtwice with 150 μl/well PBS containing 0.1% BSA and fixed for 15 min at4° C. using 100 μl/well fixation buffer (BD #554655). Aftercentrifugation, the samples were resuspended in 200 μl/well PBS 0.1% BSAcontaining DAPI to exclude dead cells for the FACS measurement. Sampleswere analyzed at BD FACS Fortessa. The results show that MCSP TCBinduced a strong and target-specific upregulation of activation markers(CD25, CD69) on CD8⁺ T cells (FIGS. 9A, B) and CD4⁺ T cells (FIGS. 9C,D) after killing.

Example 7

Cytokine Secretion by Human Effector Cells after T Cell-Killing ofMCSP-Expressing Tumor Cells Induced by MCSP TCB Antibody

Cytokine secretion by human PBMCs after T-cell killing ofMCSP-expressing MV-3 tumor cells induced by the MCSP TCB antibody wasassessed by FACS analysis of cell supernatants after the killing assay.

The same antibody was used and the killing assay was performedessentially as described above (Example 5 and 6), using an E:T ratio of10:1 and an incubation time of 24 h.

At the end of the incubation time, the plate was centrifuged for 5 minat 350×g, the supernatant transferred in a new 96-well plate and storedat −20° C. until subsequent analysis. Granzyme B, TNFα, IFN-γ, IL-2,IL-4 and IL-10 secreted into in cell supernatants were detected usingthe BD CBA Human Soluble Protein Flex Set, according to manufacturer'sinstructions on a FACS CantoII. The following kits were used: BD CBAhuman Granzyme B BD CBA human Granzyme B Flex Set #BD 560304; BD CBAhuman TNF Flex Set #BD 558273; BD CBA human IFN-γ Flex Set #BD 558269;BD CBA human IL-2 Flex Set #BD 558270; BD CBA human IL-4 Flex Set #BD558272; BD CBA human IL-10 Flex Set #BD 558274.

The results show that MCSP TCB induced secretion of IL-2, IFN-γ, TNFα,Granzyme B and IL-10 (but no IL-4) upon killing (FIG. 10, A-F).

Taken together, these examples show that the MCSP CD3 bispecificantibody

-   -   Showed a good binding to MCSP-positive A375 cells    -   Induced a strong and target-specific killing of MCSP-positive        target cell lines, and no killing of MCSP-negative cell lines    -   Induced a strong and target-specific upregulation of activation        markers (CD25, CD69) on CD8⁺ and CD4⁺ T cells after killing    -   Induced secretion of IL-2, IFN-γ, TNFα, Granzyme B and IL-10 (no        IL-4) upon killing.

Example 8 Binding of CEA TCB to CEA- and CD3-Expressing Cells

The binding of CEA TCB was tested on transfected CEA-expressing lungadenocarcinoma cells (A549-huCEA) and CD3-expressing immortalized humanand cynomolgus T lymphocyte lines (Jurkat and HSC-F, respectively). Anuntargeted TCB (SEQ ID NOs: 59, 60, 61 and 62; see example 24) was usedas control. Briefly, cells were harvested, counted, checked forviability and resuspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS0.1% BSA). 100 μl cell suspension (containing 0.2×10⁶ cells) wereincubated in round-bottom 96-well plate for 30 min at 4° C. withincreasing concentrations of the CEA TCB (61 pM-1000 nM), washed twicewith cold PBS 0.1% BSA, re-incubated for further 30 min at 4° C. withthe FITC-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgGF(ab′)2 Fragment Specific secondary antibody (Jackson Immuno ResearchLab FITC #109-096-097), washed twice with cold PBS 0.1% BSA andimmediately analyzed by FACS using a FACS CantoII or Fortessa (SoftwareFACS Diva) by gating live, PI-negative, cells. Binding curves wereobtained using GraphPadPrism5 (FIG. 11A, binding to A549 cells (EC₅₀ 6.6nM); FIG. 11B, binding to Jurkat cells; FIG. 11C, binding to HSC-Fcells).

Example 9 T Cell-Mediated Killing of CEA-Expressing Tumor Target CellsInduced by CEA TCB Antibody

T cell-mediated killing of target cells induced by CEA TCB antibody wasassessed on HPAFII (high CEA), BxPC-3 (medium CEA) and ASPC-1 (low CEA)human tumor cells. HCT-116 (CEA negative tumor cell line) and theuntargeted TCB were used as negative controls. Human PBMCs were used aseffectors and killing detected 24 h and 48 h after incubation with thebispecific antibody. Briefly, target cells were harvested withTrypsin/EDTA, washed, and plated at density of 25 000 cells/well usingflat-bottom 96-well plates. Cells were left to adhere overnight.Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and kept in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in cellincubator (37° C., 5% CO₂) until further use (no longer than 24 h). Forthe killing assay, the antibodies were added at indicated concentrations(range of 6 pM-100 nM in triplicates). PBMCs were added to target cellsat the final E:T ratio of 10:1. Target cell killing was assessed after24 h and 48 h of incubation by quantification of LDH (lactatedehydrogenase) released into cell supernatants by apoptotic/necroticcells (LDH detection kit, Roche Applied Science, #11 644 793 001).Maximal lysis of the target cells (=100%) was achieved by incubation oftarget cells with 1% Triton X-100. Minimal lysis (=0%) refers to targetcells co-incubated with effector cells without bispecific antibody. Theresults show that CEA TCB induced a strong and target-specific killingof CEA-positive target cells (FIG. 12, A-H). The EC₅₀ values related tothe killing assays, calculated using GraphPadPrism5 are given in Table8.

TABLE 8 CEA receptor copy number and EC₅₀ values (pM) for T-cellmediated killing of CEA-expressing tumor cells induced by CEA TCBantibody. CEA receptor EC50 [pM] Cell line copy number 48 h HPAFII 120000-205 000 667 BxPC-3 41 000 3785 ASPC1 3500-8000 846

Example 10

T Cell Proliferation and Activation 5 Days after CEA TCB-MediatedKilling of CEA-Expressing Tumor Target Cells

T cell proliferation and activation was detected 5 days after CEATCB-mediated killing of CEA-expressing tumor target cells assessed onHPAFII (high CEA), BxPC-3 (medium CEA) and ASPC-1 (low CEA) cells.HCT-116 (CEA negative tumor cell line) and the untargeted TCB were usedas negative controls. The experimental conditions for the proliferationassay were similar to the ones described in Example 9, but only 10 000target cells were plated per well of a 96-flat bottom well plate. Toassess T cell proliferation, freshly-isolated PBMCs were labeled usingCFSE (Sigma #21888). Briefly, CFSE stock solution was diluted to obtaina working solution of 100 μM. 90×10⁶ PBMC cells were re-suspended in 90ml pre-warmed PBS and supplemented with 90 μl of the CFSE workingsolution. Cells were mixed immediately and incubated 15 min at 37° C. 10ml of pre-warmed FCS were added to cells to stop the reaction. The cellswere centrifuged for 10 min at 400 g, re-suspended in 50 ml medium andincubated for 30 min at 37° C. After incubation, cells were washed oncewith warm medium, counted, re-suspended in medium and added to targetcells for the killing assay and subsequent measurement of cellproliferation and activation at an E:T of 10:1. Proliferation wasassessed 5 days after killing on CD4 and CD8 positive T cells byquantification of the CFSE dye dilution. CD25 expression was assessed onthe same T cell subsets using the anti-human CD25 antibody. Briefly,after centrifugation (400×g for 4 min), cells were resuspended, washedwith FACS buffer and incubated with 25 μl of the diluted CD4/CD8/CD25antibody mix for 30 min at 4° C. (APC/Cy7 anti-human CD4 #317418, APCanti-human CD8 #301014, PE/Cy7 anti-human CD25 #302612). Cells were thenwashed three times to remove the unbound antibody, and finallyresuspended in 200 μl FACS buffer containing propidium iodide (PI) toexclude dead cells for the FACS measurement. Fluorescence was measuredusing BD FACS CantoII. The results show that the CEA TCB induced astrong and target-specific proliferation of CD8⁺ and CD4⁺ T cells (FIG.13, A-D) as well as their activation as detected by up-regulation of theCD25 activation marker (FIG. 13, E-H).

Example 11

Cytokine Secretion by Human Effector Cells after T Cell-Mediated Killingof CEA-Expressing Tumor Cells Induced by CEA TCB

Cytokine secretion by human PBMCs after T cell-mediated killing ofCEA-expressing MKN45 tumor cells induced by the CEA TCB was assessed byFACS analysis (CBA kit) of cell supernatants 48 h after killing.

The experimental conditions were identical to the ones described inExample 9. At the end of the incubation time, the plate was centrifugedfor 5 min at 350×g, the supernatant transferred into a new 96-well plateand stored at −20° C. until subsequent analysis. (A) IFN-γ, (B) TNFα,(C) Granzyme B, (D) IL-2, (E) IL-6 and (F) IL-10 secreted into cellsupernatants were detected using the BD CBA Human Soluble Protein FlexSet, according to the manufacturer's instructions on a FACS CantoII. Thefollowing kits were used: BD CBA human IL-2 BD Flex Set #BD 558270; BDCBA human Granzyme B BD Flex Set #BD 560304; BD CBA human TNF Flex Set#BD 558273; BD CBA human IFN-γ Flex Set #BD 558269; BD CBA human IL-4Flex Set #BD 558272; BD CBA human IL-10 Flex Set #BD 558274.

The results show that the CEA TCB mediated killing (but not the killingmediated by untargeted TCB control) induced secretion of IFN-γ, TNFα,Granzyme B, IL-2, IL-6 and IL-10 (FIG. 14, A-F).

Example 12

T Cell-Mediated Killing of Target Cells in Presence of IncreasingConcentrations of Shed CEA (sCEA)

T cell-mediated killing of CEA-expressing tumor target cells (LS180)induced by CEA TCB antibody in presence of increasing concentrations ofshed CEA (sCEA 2.5 ng/ml-5 μg/ml) was assessed. Human PBMCs were used aseffector cells and killing detected 24 h and 48 h after incubation withthe bispecific antibody and sCEA. Briefly, target cells were harvestedwith Trypsin/EDTA, washed, and plated at density of 25 000 cells/wellusing flat-bottom 96-well plates. Cells were left to adhere overnight.Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew Falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and kept in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) in cellincubator (37° C., 5% CO₂) until further use (no longer than 24 h). Forthe killing assay, the CEA TCB antibody was used at a fixedconcentration of 1 nM and sCEA was spiked into the experiment at aconcentration range of 2.5 ng-5 μg/ml. PBMCs were added to target cellsat the final E:T ratio of 10:1. Target cell killing was assessed after24 h and 48 h of incubation by quantification of LDH (lactatedehydrogenase) released into cell supernatants by apoptotic/necroticcells (LDH detection kit, Roche Applied Science, #11 644 793 001).Maximal lysis of the target cells (=100%) was achieved by incubation oftarget cells with 1% Triton X-100. Minimal lysis (=0%) refers to targetcells co-incubated with effector cells without bispecific antibody. Thekilling mediated by CEA TCB in absence of sCEA was set at 100% and thekilling obtained in presence of increasing concentrations of sCEA wasnormalized to it. Results show that sCEA had only a minor impact on CEATCB-mediated killing of CEA-expressing target cells (FIG. 15A, B). Noeffect on T cell killing was detected up to 0.2 μg/ml of sCEA. The sCEAconcentrations above 0.2 μg/ml had only a minor impact on overallkilling (10-50% reduction).

Example 13 T Cell-Mediated Killing of Target Cells Using Human andCynomolgus PBMCs as Effector Cells

T cell-mediated killing of A549 (lung adenocarcinoma) cellsoverexpressing human CEA (A549-hCEA), assessed 21 h and 40 h afterincubation with CEA TCB antibody and human PBMCs or cynomolgus PBMCs aseffector cells was assessed. Briefly, target cells were harvested withTrypsin/EDTA, washed, and plated at density of 25 000 cells/well usingflat-bottom 96-well plates. Cells were left to adhere for several hours.Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors or healthy cynomolgus monkey. For thelater, a 90% Histopaque-PBS density gradient was used. Fresh blood wasdiluted with sterile PBS and layered over Histopaque gradient (Sigma,#H8889). After centrifugation (450×g, 30 minutes, room temperature forhuman PBMCs, respective 520×g, 30 min, room temperature for cynomolgusPBMCs), the plasma above the PBMC-containing interphase was discardedand PBMCs transferred in a new Falcon tube subsequently filled with 50ml of PBS. The mixture was centrifuged (400×g, 10 minutes, roomtemperature), the supernatant discarded and the PBMC pellet washed twicewith sterile PBS (centrifugation steps 350×g, 10 minutes). For thepreparation of the cynomolgus PBMCs, an additional low-speedcentrifugation step was performed at 150×g for 15 min. The resultingPBMC population was counted automatically (ViCell) and kept in RPMI1640medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302)in cell incubator (37° C., 5% CO₂) until further use (up to 4h). For thekilling assay, the antibodies were added at indicated concentrations(range of 6 pM-100 nM in triplicates). PBMCs were added to target cellsat the final E:T ratio of 10:1. Target cell killing was assessed after21 h and 40 h of incubation by quantification of LDH (lactatedehydrogenase) released into cell supernatants by apoptotic/necroticcells (LDH detection kit, Roche Applied Science, #11 644 793 001).Maximal lysis of the target cells (=100%) was achieved by incubation oftarget cells with 1% Triton X-100. Minimal lysis (=0%) refers to targetcells co-incubated with effector cells without bispecific antibody.Results show that CEA TCB mediates target-specific killing ofCEA-positive target cells using both human (FIG. 16, A, C) andcynomolgus (FIG. 16, B, D) effector cells (PBMCs). The EC₅₀ valuesrelated to 40 h of killing, calculated using GraphPadPrism5 are 306 pMfor human PBMCs and 102 pM for cynomolgus PBMCs.

Example 14 T Cell-Mediated Killing of CEA-Expressing Human ColorectalCancer Cell Lines Induced by CEA TCB Antibody

T cell-mediated killing of CEA-expressing human colorectal cancer celllines 48 h after incubation with human PBMCs and CEA TCB antibody at 0.8nM, 4 nM and 20 nM was assessed. Briefly, PBMCs were isolated fromleukocyte cones obtained from single healthy donors. Cells were dilutedwith PBS (1:10) and layered on Lymphoprep in 50 mL Falcon tubes. Aftercentrifugation (1800 rpm for 25 min), the PBMC layer was withdrawn fromthe interface and washed 4× with PBS. PBMCs were counted, frozen in 10%DMSO in FCS under controlled-rate freezing conditions at 40×10⁶ cells/mLand stored in liquid nitrogen until further use. For the T-cell killingassay, tumor cells were plated directly into 96-well plates from frozenstocks. Cells were warmed quickly and transferred immediately intopre-warmed medium, centrifuged, and re-suspended in complete medium(DMEM, Iscoves or RPMI-1640, all supplemented with 10% FCS and 1%penicillin/streptomycin) and plated at a density of 2.5×10⁴ cells/well.Plates were then incubated at 37° C. in a humidified 10% CO₂ incubatorand medium replaced the next day by 100 μL of RPMI 2% FCS with 1%glutamine and 50 μL CEA TCB (final concentrations ranging from 6.4 to20000 pM, 1:5 titration steps, in duplicate wells for each condition).Fresh-thawed PBMCs were used for the assay (thawed from frozen vialswithin 2 hours of the assay start) and 50 μL (3×10⁵) was added to eachwell to give an effector:target (E:T) ratio of 10:1. Triton X100 (50 μLof 4%) was added to 150 μL of target cells to obtain maximum releasevalues. Plates were incubated at 37° C. for 48 h and the killingactivity determined using the Lactose Dehydrogenase CytotoxicityDetection Kit (Roche) in accordance with the manufacturer'sinstructions. Percentage of specific cell lysis was calculated as[sample release−spontaneous release]/[maximum release−spontaneousrelease]×100. FIG. 17, A-C shows the correlation between CEA expression(receptor copy number quantified using QIFIKIT, see below) and % killingfor 31 colorectal cancer cell lines (listed on x axis). FIG. 17, D showsthe correlation between CEA expression and % specific lysis at 20 nM ofCEA TCB (Spearman correlation=0.7289, p<0.0001, n=31), indicating thattumor cells displaying high CEA receptor copy numbers (>50 000) areefficiently lysed by CEA TCB whereas a cluster of cells displaying lowCEA receptor copy numbers (≤10 000) are not being lysed by CEA TCB underthe same experimental conditions. FIG. 17, E shows the correlationbetween CEA expression and EC₅₀ of CEA TCB. Although the correlation isnot statistically significant (Spearman correlation=−0.3994, p=0.1006,R²=0.1358) the graph clearly shows a pattern of better CEA TCB potency(i.e. lower EC₅₀ values) on tumor cell lines expressing high CEAreceptor copy numbers.

For the analysis of CEA surface expression on cancer cell lines, theQifikit (DakoCytomation, Glostrup, Denmark) was used to calibrate thefluorescent signals and determine the number of binding sites per cell.Cells were incubated on ice for 30 min with a mouse anti-human CEACAM5monoclonal antibody (0.5 μg for 5×105 cells, clone: CI-P83-1, sc-23928,Santa Cruz), washed twice with PBS1X-BSA 0.1% followed by a 45 minincubation with polyclonal fluorescein isothiocyanate-conjugated goatanti-mouse antibody provided with the Qifikit. Dead cells were excludedfrom the analysis using 4′,6-diamidino-2-phenylindole (DAPI) staining.Samples were analysed on a CyAn™ ADP Analyzer (Beckman Coulter). Allmean fluorescence intensities (MFIs) were obtained after data analysesusing Summit 4.3 software. These MFIs were used to determine therelative number of antibody binding sites on the cell lines (named asCEA copy number on the results) using the equation obtained from thecalibration curve (Qifikit calibration beads).

The colorectal cancer cell lines used for the T-cell killing assays andCEA surface expression quantification were seeded from cryovials. Themethod used to maintain the frozen stock was as described in Bracht etal. (Bracht et al. (2010), Br J Cancer 103, 340-346).

Example 15

In Vivo Anti-Tumor Efficacy of CEA TCB in a LS174T-Fluc2 Human ColonCarcinoma Co-Grafted with Human PBMC (E:T Ratio 5:1)

NOG (NOD/Shi-scid/IL-2Rγnull) mice (n=12) were injected subcutaneouslywith 1×10⁶LS174T-fluc2 cells pre-mixed with human PBMC in a total volumeof 100 μl in PBS, E:T ratio 5:1. LS174T-fluc2 cells have been engineeredto express luciferase, which allows monitoring tumor progression bybioluminescence (BLI) in a non-invasive and highly sensitive manner. Toassess early and delayed treatment effects, mice received bi-weekly i.v.injections of either 0.5 or 2.5 mg/kg of the CEA TCB starting at day 1(early treatment) or day 7 (delayed treatment) after tumor cell/PBMCsco-grafting s.c. As a control, one group of mice received bi-weekly i.v.injections of 2.5 mg/kg of a control TCB that had the same format as CEATCB (in this case the MCSP TCB served as untargeted control sinceLS174T-fluc2 cells do not express MCSP), and an extra control groupreceived only PBS (vehicle) starting at day 1. Tumor volume was measuredonce a week by digital caliper. Furthermore, mice were injected i.p.once weekly with D-Luciferin and the bioluminescent light emission ofliving tumor cells was measured with IVIS Spectrum (Perkin Elmer).Treatment was administered until 19 days after tumor cell inoculation,which corresponds to the day of study termination. The results of theexperiment are shown in FIG. 18A-D. Results show average and SEM from 12mice of tumor volume measured by caliper (A and C) and bybioluminescence (Total Flux, B and D) in the different study groups ((A,B) early treatment, (C, D) delayed treatment).

Example 16

In Vivo Anti-Tumor Efficacy of CEA TCB in a LS174T-Fluc2 Human ColonCarcinoma Co-Grafted with Human PBMC (E:T Ratio 1:1)

NOG (NOD/Shi-scid/IL-2Rγnull) mice (n=10) were injected subcutaneouslywith 1×10⁶ LS174T-fluc2 cells (see Example 15) pre-mixed with human PBMCin a total volume of 100 μl in PBS, E:T ratio 1:1. To assess early anddelayed treatment effects, mice received bi-weekly i.v. injections of2.5 mg/kg of the CEA TCB starting at day 1 (early treatment) or day 7(delayed treatment) after tumor cell inoculation. As control, one groupof mice received bi-weekly i.v. injections of 2.5 mg/kg of the MCSP TCB(see also Example 15), and an extra control group received only PBS(vehicle) starting at day 1. Tumor volume was measured once weekly bydigital caliper. Furthermore, mice were injected i.p. once weekly withD-Luciferin and the bioluminescent light emission of living tumor cellswas measured with IVIS Spectrum (Perkin Elmer). Treatment wasadministered until 23 days after tumor cell inoculation, whichcorresponds to the day of study termination. The results of theexperiment are shown in FIG. 19. Results show average and SEM of tumorvolume measured by caliper (A) as well as by bioluminescence (B) in thedifferent study groups (n=10).

Example 17

In Vivo Efficacy of Murinized CEA TCB in a Panco2-huCEA Orthotopic TumorModel in Immunocompetent huCD3E/huCEA Transgenic Mice

huCD3c/huCEA transgenic mice (n=10) received an intra-pancreaticinjection of 2×10⁵ Panco2-huCEA cells in a total volume of 10 μl in PBS.As murine cells do not express CEA, the murine pancreatic carcinoma cellline Panco2 was engineered to overexpress human CEA as the targetantigen for the CEA TCB. Mice were injected twice weekly i.v. with 0.5mg/kg of the murinized CEA TCB or PBS as a control group (vehicle) andsurvival was monitored. Animals were controlled daily for clinicalsymptoms and detection of adverse effects. Termination criteria foranimals were visible sickness: scruffy fur, arched back, breathingproblems, impaired locomotion. The result as overall survival is shownin FIG. 20. Result shows percent of surviving animals per time point.The significance of the treatment group to the PBS control group wascompared using a paired Student t test (p=0.078).

Example 18 Affinity of the CEA TCB to CEA and CD3 by Surface PlasmonResonance (SPR)

Surface plasmon resonance (SPR) experiments were performed on a BiacoreT100 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

For affinity measurements CEA TCB was captured on a CM5 sensorchipsurface with immobilized anti human Fab (GE Healthcare #28-9583-25).Capture IgG was coupled to the sensorchip surface by directimmobilization of around 10,000 resonance units (RU) at pH 5.0 using thestandard amine coupling kit (Biacore, Freiburg/Germany).

To analyze the interaction to human CD3ε stalk-Fc(knob)-Avi/CD3δ-stalk-Fc(hole) (SEQ ID NOs 120 and 121, respectively),CEA TCB was captured for 30 s at 50 nM with 10 μl/min. CD3ε/CD3δ waspassed at a concentration of 0.68-500 nM with a flowrate of 30 μl/minthrough the flow cells over 360 s. The dissociation was monitored for360 s.

The K_(D) value of the interaction between CEA TCB and the recombinanttumor target antigen human NAB A-avi-his (containing the B3 domain ofhuman CEA (CEACAM5) surrounded by the N, A1 and A2 domain of humanCEACAM1 with a C-terminal avi 6his tag; see SEQ ID NO: 119) wasdetermined by capturing the TCB molecule for 40 s at 10 μl/min. Theantigen was flown over the flow cell for 240 s in a concentration rangefrom 0.68 to 500 nM at a flow rate of 30 μl/min. The dissociation wasmeasured over 240 s.

Bulk refractive index differences were corrected for by subtracting theresponse obtained on a reference flow cell. Here, the antigens wereflown over a surface with immobilized anti-human Fab antibody but onwhich HBS-EP has been injected rather than CEA.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration. The half-life (t_(1/2))of the interaction was calculated using following formula: t_(1/2)=ln2/k_(off).

The CEA TCB binds to the tumor target and CD3ε/CD3δ in the nM-range withK_(D) values of 62 nM for the human NABA and 75.3 nM for the humanCD3ε/CD3δ. The half-life of the monovalent binding to NABA is 5.3minutes, the half-life of the binding to CD3ε/CD3δ is 5.7 minutes. Thekinetic values are summarized in Table 9.

TABLE 9 Affinity of CEA TCB to human NABA and human CD3ε/CD3δ (T = 25°C). Antigen TCB k_(on) [1/Ms] k_(off) [1/s] t_(1/2) [min] K_(D) [nM]Human CEA TCB 3.49 × 10⁴ 2.18 × 10⁻³ 5.3 62.4 NABA Human CD3ε/CD3δ CEATCB 2.69 × 10⁴ 2.03 × 10⁻³ 5.7 75.3

Example 19 Affinity of the MSCP TCB to MCSP and CD3 by Surface PlasmonResonance (SPR)

Surface plasmon resonance (SPR) experiments were performed on a BiacoreT100 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

For affinity measurements MCSP TCB was captured on a CM5 sensorchipsurface with immobilized anti human Fab (GE Healthcare #28-9583-25).Capture IgG was coupled to the sensorchip surface by directimmobilization of around 7,500 resonance units (RU) at pH 5.0 using thestandard amine coupling kit (Biacore, Freiburg/Germany). MCSP TCB wascaptured for 60 s at 30 nM with 10 μl/min. Human and cynomolgus MCSP D3(see SEQ ID NOs 118 and 117, respectively) were passed at aconcentration of 0.024-50 nM with a flowrate of 30 μl/min through theflow cells over 90 s. The concentration range for human and cynomolgusCD3c stalk-Fc (knob)-Avi/CD3δ-stalk-Fc(hole) was 1.17-600 nM. Since theinteraction with murine MCSP D3 (SEQ ID NO: 122) was expected to be weakthe concentration range for this antigen was chosen between 3.9 and 500nM. The dissociation for all interactions was monitored for 120 s. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on a reference flow cell. Here, the antigens wereflown over a surface with immobilized anti-human Fab antibody but onwhich HBS-EP has been injected rather than MCSP TCB.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration. The interaction for theMCSP TCB with the murine MCSP D3 was determined in steady state. Thehalf-life (t_(1/2)) of the interaction was calculated using followingformular: t_(1/2)=ln 2/k_(off).

The MCSP TCB binds to the tumor target in pM-range with K_(D) values of0.15 nM for the human and 0.12 nM for the cynomolgus antigen.Recombinant CD3ε/CD3δ is bound by the MCSP TCB with a K_(D) value of 78nM (human) and 104 nM (cynomolgus). The half-life of the monovalentbinding is up to 260 minutes for the tumor target and 2.9 minutes forthe CD3e/CD3d. Upon affinity maturation the MCSP antibody obtained somebinding to recombinant murine MCSP D3. K_(D) value for this interactionis in mM range (1.6 mM). The kinetic values are summarized in Table 10.

TABLE 10 Affinity of MCSP TCB to the human, cynomolgus and murine MCSPD3 and human and cynomolgus CD3ε/CD3δ (T = 25° C.). k_(on) [1/Ms]k_(off) [1/s] t_(1/2) [min] K_(D) [nM] Human MCSP D3 3.89 × 10⁵ 5.63 ×10−⁵ 205 0.15 Cynomolgus MCSP D3 3.70 × 10⁵ 4.39 × 10−⁵ 263 0.12 MurineMCSP D3 nd nd nd 1570*    Human CD3ε/CD3δ 4.99 × 10⁴ 3.92 × 10⁻³ 2.978.7  Cynomolgus CD3ε/ 4.61 × 10⁴ 4.78 × 10⁻³ 2.4 104    CD3δ*determined by steady state measurement

Example 20 Thermal Stability of CEA TCB

Thermal stability of the CEA TCB was monitored by Dynamic LightScattering (DLS). 30 μg of filtered protein sample with a proteinconcentration of 0.5 mg/ml was applied in duplicate to a Dynapro platereader (Wyatt Technology Corporation; USA). The temperature was rampedfrom 25 to 75° C. at 0.05° C./min, with the radius and total scatteringintensity being collected. The result is shown in FIG. 21. Theaggregation temperature of the CEA TCB was measured at 55° C.

Example 21 Thermal Stability of MCSP TCB

Thermal stability of the MCSP TCB was monitored by Dynamic LightScattering (DLS). 30 μg of filtered protein sample with a proteinconcentration of 0.5 mg/ml was applied in duplicate to a Dynapro platereader (Wyatt Technology Corporation; USA). The temperature was rampedfrom 25 to 75° C. at 0.05° C./min, with the radius and total scatteringintensity being collected.

The result is shown in FIG. 22. The aggregation temperature of the MCSPTCB was measured at 55° C.

Example 22 T Cell-Mediated Killing of MCSP-Expressing Tumor Target CellsInduced by MCSP TCB and MCSP 1+1 CrossMab Antibodies

T cell-mediated killing of target cells induced by MCSP TCB and MCSP 1+1CrossMab TCB (a T cell activating bispecific antibody having the sameCD3 and MCSP binding sequences as the MCSP TCB, with the molecularformat shown in FIG. 1D) antibodies was assessed on A375 (high MCSP),MV-3 (medium MCSP) and HCT-116 (low MCSP) tumor target cells. LS180(MCSP negative tumor cell line) was used as negative control. Tumor cellkilling was assessed 24 h and 48 h post incubation of target cells withthe antibodies and effector cells (human PBMCs). Briefly, target cellswere harvested with Trypsin/EDTA, washed, and plated at density of 25000 cells/well using flat-bottom 96-well plates. Cells were left toadhere overnight. Peripheral blood mononuclear cells (PBMCs) wereprepared by Histopaque density centrifugation of enriched lymphocytepreparations (buffy coats) obtained from healthy human donors. Freshblood was diluted with sterile PBS and layered over Histopaque gradient(Sigma, #H8889). After centrifugation (450×g, 30 minutes, roomtemperature), the plasma above the PBMC-containing interphase wasdiscarded and PBMCs transferred in a new Falcon tube subsequently filledwith 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, roomtemperature), the supernatant discarded and the PBMC pellet washed twicewith sterile PBS (centrifugation steps 350×g, 10 minutes). The resultingPBMC population was counted automatically (ViCell) and kept in RPMI1640medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302)in cell incubator (37° C., 5% CO₂) until further use (no longer than 24h). For the killing assay, the antibodies were added at indicatedconcentrations (range of 0.01 pM-10 nM in triplicates). PBMCs were addedto target cells at the final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation by quantification of LDH(lactate dehydrogenase) released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific antibody. The results show that MCSP TCB antibody is morepotent than the MCSP 1+1 CrossMab TCB as it induced stronger killing ofMCSP-positive target cells at both time points and on all tumor targetcells (FIG. 23A-H). The EC₅₀ values related to killing assays,calculated using GraphPadPrism5, are given in Table 11.

TABLE 11 MCSP receptor copy number and EC₅₀ values (pM) for T-cellmediated killing of MCSP-expressing tumor cells induced by MCSP TCBantibody (n.d. = not determined). MCSP receptor EC50 [pM] EC50 [pM] Cellline copy number 24 h 48 h A375 387 058 0.1 n.d. MV-3 260 000 1.0  0.7HCT-116 36770 ~6.2e−008 ~0.09 LS180 negative ~764 n.d.

Example 23

CD25 and CD69 Upregulation on CD8⁺ and CD4⁺ Effector Cells after TCell-Mediated Killing of MCSP-Expressing Tumor Cells Induced by MCSP TCBand MCSP 1+1 CrossMab Antibodies

Activation of CD8⁺ and CD4⁺ T cells after T-cell killing ofMCSP-expressing tumor cells (A375 and MV-3) mediated by the MCSP TCB andMCSP 1+1 CrossMab antibodies was assessed by FACS analysis usingantibodies recognizing T cell activation markers CD25 (late activationmarker) and CD69 (early activation marker). The antibody and the killingassay conditions were essentially as described above (Example 22), usingthe same antibody concentration range (0.01 pM-10 nM in triplicates),E:T ratio 10:1 and an incubation time of 48 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 350×g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (FITC anti-human CD8, BD#555634), CD4 (PECy7 anti-human CD4, BD #557852), CD69 (PE anti-humanCD69, Biolegend #310906) and CD25 (APC anti-human CD25, BD #555434) wasperformed according to the suppliers' indications. Cells were washedtwice with 150 μl/well PBS containing 0.1% BSA and fixed for 15 min at4° C. using 100 μl/well fixation buffer (BD #554655). Aftercentrifugation, the samples were resuspended in 200 μl/well PBS 0.1% BSAcontaining DAPI to exclude dead cells for the FACS measurement. Sampleswere analyzed at BD FACS Fortessa. The results show that MCSP TCBinduced a strong and target-specific upregulation of activation markers(CD25, CD69) on CD8⁺ T cells (FIGS. 24A, B (for A375 cells) and E, F(for MV-3 cells)) and CD4⁺ T cells (FIGS. 24C, D (for A375 cells) and G,H (for MV-3 cells)) after killing. As for the killing results, theactivation of T cells was stronger with MCSP TCB than with MCSP 1+1CrossMab.

Example 24 Preparation of DP47 GS TCB (2+1 Crossfab-IgG P329G LALAInverted=“Untargeted TCB”) Containing DP47 GS as Non Binding Antibodyand Humanized CH2527 as Anti CD3 Antibody

The “untargeted TCB” was used as a control in the above experiments. Thebispecific antibody engages CD3ε but does not bind to any other antigenand therefore cannot crosslink T cells to any target cells (andsubsequently cannot induce any killing). It was therefore used asnegative control in the assays to monitor any unspecific T cellactivation.

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the constant heavy chain or the constantlight chain pre-inserted into the respective recipient mammalianexpression vector. The antibody expression is driven by an MPSV promoterand carries a synthetic polyA signal sequence at the 3′ end of the CDS.In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293 EBNA cells with themammalian expression vectors using polyethylenimine (PEI). The cellswere transfected with the corresponding expression vectors in a 1:2:1:1ratio (“vector heavy chain Fc(hole)”:“vector light chain”:“vector lightchain Crossfab”: “vector heavy chain Fc(knob)-FabCrossfab”).

For transfection HEK293 EBNA cells were cultivated in suspensionserum-free in CD CHO culture medium. For the production in 500 ml shakeflask 400 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection cells were centrifuged for 5 min at210×g, supernatant is replaced by pre-warmed 20 ml CD CHO medium.Expression vectors were mixed in 20 ml CD CHO medium to a final amountof 200 μg DNA. After addition of 540 μl PEI solution the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 500 ml shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. After the incubation time 160ml F17 medium was added and cell were cultivated for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed 1 (Lonza) was added.After 7 days cultivation supernatant was collected for purification bycentrifugation for 15 min at 210×g, the solution was sterile filtered(0.22 μm filter) and sodium azide in a final concentration of 0.01% w/vwas added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using Protein A. Supernatant was loaded on aHiTrap Protein A HP column (CV=5 mL, GE Healthcare) equilibrated with 40ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,pH 7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, pH 7.5. Target protein was eluted during a gradient over 20column volumes from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solutionwas neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Targetprotein was concentrated and filtrated prior loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of molecules were analyzed by CE-SDSanalyses in the presence and absence of a reducing agent. The CaliperLabChip GXII system (Caliper lifescience) was used according to themanufacturer's instruction. 2 μg sample was used for analyses.

The aggregate content of antibody samples was analyzed using a TSKgelG3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C.

TABLE 12 Summary production and purification of DP47 GS TCB. Aggregateafter 1^(st) Titer Yield purification HMW LMW Monomer Construct [mg/l][mg/l] step [%] [%] [%] [%] DP47 GS TCB 103.7 8.04 8 2.3 6.9 91.8

FIG. 25 and Table 13 show CE-SDS analyses of the DP47 GS TCB (2+1Crossfab-IgG P329G LALA inverted) containing DP47 GS as non-bindingantibody and humanized CH2527 as anti-CD3 antibody. (SEQ ID NOs: 59, 60,61 and 62).

TABLE 13 CE-SDS analyses of DP47 GS TCB. Peak kDa Corresponding ChainDP47 GS TCB 1 165.22 Molecule with 2 missing light non reduced (A)chains 2 181.35 Molecule with 1 missing light chain 3 190.58 Correctmolecule without N- linked glycosylation 4 198.98 Correct molecule DP47GS TCB 1 27.86 Light chain DP47 GS reduced (B) 2 35.74 Light chainhuCH2527 3 63.57 Fc(hole) 4 93.02 Fc(knob)

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A T cell activating bispecific antigen binding molecule comprising(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and at leastone light chain CDR selected from the group of SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NO: 10; (ii) a second antigen binding moiety which is a Fabmolecule capable of specific binding to a target cell antigen.
 2. The Tcell activating bispecific antigen binding molecule of claim 1, whereinthe first antigen binding moiety comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to an amino acid sequence selected from thegroup of: SEQ ID NO: 3, SEQ ID NO: 32 and SEQ ID NO: 33 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence selected from the group of: SEQ ID NO: 7 and SEQ ID NO:
 31. 3.The T cell activating bispecific antigen binding molecule of claim 1,wherein the first antigen binding moiety comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 3 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:
 7. 4. The T cellactivating bispecific antigen binding molecule of claim 1, wherein thesecond antigen binding moiety is capable of specific binding toCarcinoembryonic Antigen (CEA) and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 and atleast one light chain CDR selected from the group of SEQ ID NO: 28, SEQID NO: 29 and SEQ ID NO:
 30. 5. The T cell activating bispecific antigenbinding molecule of claim 4, wherein the second antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 23 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:
 27. 6. The T cell activating bispecific antigen bindingmolecule of claim 1, wherein the second antigen binding moiety iscapable of specific binding to Melanoma-associated Chondroitin SulfateProteoglycan (MCSP) and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO:
 50. 7. The T cell activating bispecific antigenbinding molecule of claim 6, wherein the second antigen binding moietycomprises at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15and SEQ ID NO: 16 and at least one light chain CDR selected from thegroup of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO:
 20. 8. The T cellactivating bispecific antigen binding molecule of claim 6, wherein thesecond antigen binding moiety is capable of specific binding to MCSP andcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group of SEQ IDNO: 13, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from the group of SEQ ID NO: 17, SEQ ID NO: 43,SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO:
 51. 9. The T cell activatingbispecific antigen binding molecule of claim 8, wherein the secondantigen binding moiety comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:
 17. 10. The T cell activating bispecificantigen binding molecule of claim 1, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions of the Fab light chain and the Fab heavy chain areexchanged.
 11. The T cell activating bispecific antigen binding moleculeof claim 10, wherein the first antigen binding moiety is a crossover Fabmolecule wherein the constant regions of the Fab light chain and the Fabheavy chain are exchanged.
 12. The T cell activating bispecific antigenbinding molecule of claim 1, wherein the second antigen binding moietyis a conventional Fab molecule.
 13. The T cell activating bispecificantigen binding molecule of claim 1, comprising not more than oneantigen binding moiety capable of specific binding to CD3.
 14. The Tcell activating bispecific antigen binding molecule of claim 1,comprising a third antigen binding moiety which is a Fab moleculecapable of specific binding to a target cell antigen.
 15. The T cellactivating bispecific antigen binding molecule of claim 14, wherein thethird antigen binding moiety is a conventional Fab molecule.
 16. The Tcell activating bispecific antigen binding molecule of claim 14, whereinthe third antigen binding moiety is identical to the second antigenbinding moiety.
 17. The T cell activating bispecific antigen bindingmolecule of claim 16, wherein the third antigen binding moiety is anantigen binding moiety capable of specific binding to CEA and comprisesat least one heavy chain complementarity determining region (CDR)selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 andSEQ ID NO: 26 and at least one light chain CDR selected from the groupof SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:
 30. 18. The T cellactivating bispecific antigen binding molecule of claim 16, wherein thethird antigen binding moiety is an antigen binding moiety capable ofspecific binding to MCSP and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 40 and at least onelight chain CDR selected from the group of SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 20, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO:49 and SEQ ID NO:
 50. 19. The T cell activating bispecific antigenbinding molecule of claim 1, wherein the first and the second antigenbinding moiety are fused to each other, optionally via a peptide linker.20. The T cell activating bispecific antigen binding molecule of claim1, wherein the second antigen binding moiety is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding moiety.
 21. The T cell activating bispecificantigen binding molecule of claim 1, wherein the first antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.22. The T cell activating bispecific antigen binding molecule of claim20, wherein the Fab light chain of the first antigen binding moiety andthe Fab light chain of the second antigen binding moiety are fused toeach other, optionally via a peptide linker.
 23. The T cell activatingbispecific antigen binding molecule of claim 1, additionally comprising(iii) an Fc domain composed of a first and a second subunit capable ofstable association.
 24. The T cell activating bispecific antigen bindingmolecule of claim 23, wherein the second antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the firstor the second subunit of the Fc domain.
 25. The T cell activatingbispecific antigen binding molecule of claim 23, wherein the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or second subunit of the Fc domain. 26.The T cell activating bispecific antigen binding molecule of claim 23,wherein the first and the second antigen binding moiety are each fusedat the C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain.
 27. The T cell activating bispecific antigenbinding molecule of claim 23, wherein a third antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or second subunit of the Fc domain.
 28. The T cell activatingbispecific antigen binding molecule of claim 27, wherein the second andthe third antigen binding moiety are each fused at the C-terminus of theFab heavy chain to the N-terminus of one of the subunits of the Fcdomain, and the first antigen binding moiety is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety.
 29. The T cell activating bispecificantigen binding molecule of claim 27, wherein the first and the thirdantigen binding moiety are each fused at the C-terminus of the Fab heavychain to the N-terminus of one of the subunits of the Fc domain, and thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety.
 30. The T cell activating bispecific antigenbinding molecule of claim 29, wherein the first and the third antigenbinding moiety and the Fc domain are part of an immunoglobulin molecule,particularly an IgG class immunoglobulin.
 31. A T cell activatingbispecific antigen binding molecule comprising (i) a first antigenbinding moiety which is a Fab molecule capable of specific binding toCD3, comprising the heavy chain complementarity determining region (CDR)1 of SEQ ID NO: 4, the heavy chain CDR 2 of SEQ ID NO: 5, the heavychain CDR 3 of SEQ ID NO: 6, the light chain CDR 1 of SEQ ID NO: 8, thelight chain CDR 2 of SEQ ID NO: 9 and the light chain CDR 3 of SEQ IDNO: 10, wherein the first antigen binding moiety is a crossover Fabmolecule wherein either the variable or the constant regions,particularly the constant regions, of the Fab light chain and the Fabheavy chain are exchanged; (ii) a second and a third antigen bindingmoiety each of which is a Fab molecule capable of specific binding toCEA comprising the heavy chain CDR 1 of SEQ ID NO: 24, the heavy chainCDR 2 of SEQ ID NO: 25, the heavy chain CDR 3 of SEQ ID NO: 26, thelight chain CDR 1 of SEQ ID NO: 28, the light chain CDR 2 of SEQ ID NO:29 and the light chain CDR3 of SEQ ID NO:
 30. 32. The T cell activatingbispecific antigen binding molecule of claim 31, comprising (i) a firstantigen binding moiety which is a Fab molecule capable of specificbinding to CD3 comprising a heavy chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 3 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7, wherein the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged; (ii) a second and a third antigenbinding moiety each of which is a Fab molecule capable of specificbinding to CEA comprising heavy chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 23 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:
 27. 33. A T cell activating bispecific antigenbinding molecule comprising (i) a first antigen binding moiety which isa Fab molecule capable of specific binding to CD3, comprising the heavychain complementarity determining region (CDR) 1 of SEQ ID NO: 4, theheavy chain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO:6, the light chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQID NO: 9 and the light chain CDR 3 of SEQ ID NO: 10, wherein the firstantigen binding moiety is a crossover Fab molecule wherein either thevariable or the constant regions, particularly the constant regions, ofthe Fab light chain and the Fab heavy chain are exchanged; (ii) a secondand a third antigen binding moiety each of which is a Fab moleculecapable of specific binding to MCSP comprising the heavy chain CDR 1 ofSEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chainCDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 18, thelight chain CDR 2 of SEQ ID NO: 19 and the light chain CDR3 of SEQ IDNO:
 20. 34. The T cell activating bispecific antigen binding molecule ofclaim 33, comprising (i) a first antigen binding moiety which is a Fabmolecule capable of specific binding to CD3 comprising a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 3 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, wherein the firstantigen binding moiety is a crossover Fab molecule wherein either thevariable or the constant regions, particularly the constant regions, ofthe Fab light chain and the Fab heavy chain are exchanged; (ii) a secondand a third antigen binding moiety each of which is a Fab moleculecapable of specific binding to MCSP comprising a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 13 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:
 17. 35. The T cellactivating bispecific antigen binding molecule of claim 31, furthercomprising (iii) an Fc domain composed of a first and a second subunitcapable of stable association, wherein the second antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding moiety, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain, and wherein thethird antigen binding moiety is fused at the C-terminus of the Fab heavychain to the N-terminus of the second subunit of the Fc domain.
 36. TheT cell activating bispecific antigen binding molecule of claim 23,wherein the Fc domain is an IgG, specifically an IgG₁ or IgG₄, Fcdomain.
 37. The T cell activating bispecific antigen binding molecule ofclaim 23, wherein the Fc domain is a human Fc domain.
 38. The T cellactivating bispecific antigen binding molecule of claim 23, wherein theFc domain comprises a modification promoting the association of thefirst and the second subunit of the Fc domain.
 39. The T cell activatingbispecific antigen binding molecule of claim 38, wherein in the CH3domain of the first subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance within the CH3 domain of the firstsubunit which is positionable in a cavity within the CH3 domain of thesecond subunit, and in the CH3 domain of the second subunit of the Fcdomain an amino acid residue is replaced with an amino acid residuehaving a smaller side chain volume, thereby generating a cavity withinthe CH3 domain of the second subunit within which the protuberancewithin the CH3 domain of the first subunit is positionable.
 40. The Tcell activating bispecific antigen binding molecule of claim 23, whereinthe Fc domain exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. 41.The T cell activating bispecific antigen binding molecule of claim 23,wherein the Fc domain comprises one or more amino acid substitution thatreduces binding to an Fc receptor and/or effector function.
 42. The Tcell activating bispecific antigen binding molecule of claim 41, whereinsaid one or more amino acid substitution is at one or more positionselected from the group of L234, L235, and P329 (Kabat numbering). 43.The T cell activating bispecific antigen binding molecule of claim 42,wherein each subunit of the Fc domain comprises three amino acidsubstitutions that reduce binding to an activating Fc receptor and/oreffector function wherein said amino acid substitutions are L234A, L235Aand P329G.
 44. The T cell activating bispecific antigen binding moleculeof claim 40, wherein the Fc receptor is an Fey receptor.
 45. The T cellactivating bispecific antigen binding molecule of claim 40, wherein theeffector function is antibody-dependent cell-mediated cytotoxicity(ADCC).
 46. An isolated polynucleotide encoding the T cell activatingbispecific antigen binding molecule of claim 1 or a fragment thereof.47. An isolated polynucleotide encoding the T cell activating bispecificantigen binding molecule of claim 31 or a fragment thereof.
 48. Anisolated polynucleotide encoding the T cell activating bispecificantigen binding molecule of claim 33 or a fragment thereof.
 49. Apolypeptide encoded by the polynucleotide of claim
 46. 50. A vector,particularly an expression vector, comprising the polynucleotide ofclaim
 46. 51. A host cell comprising the vector of claim
 50. 52. Amethod of producing the T cell activating bispecific antigen bindingmolecule capable of specific binding to CD3 and a target cell antigen,comprising the steps of a) culturing the host cell of claim 51 underconditions suitable for the expression of the T cell activatingbispecific antigen binding molecule and b) recovering the T cellactivating bispecific antigen binding molecule.
 53. A T cell activatingbispecific antigen binding molecule produced by the method of claim 52.54. A pharmaceutical composition comprising the T cell activatingbispecific antigen binding molecule of claim 1 and a pharmaceuticallyacceptable carrier.
 55. A pharmaceutical composition comprising the Tcell activating bispecific antigen binding molecule of claim 31 and apharmaceutically acceptable carrier.
 56. A pharmaceutical compositioncomprising the T cell activating bispecific antigen binding molecule ofclaim 33 and a pharmaceutically acceptable carrier.
 57. A pharmaceuticalcomposition comprising the T cell activating bispecific antigen bindingmolecule of claim 53 and a pharmaceutically acceptable carrier.
 58. TheT cell activating bispecific antigen binding molecule of claim 1 for useas a medicament.
 59. The T cell activating bispecific antigen bindingmolecule of claim 31 for use as a medicament.
 60. The T cell activatingbispecific antigen binding molecule of claim 33 for use as a medicament.61. The T cell activating bispecific antigen binding molecule of claim53 for use as a medicament.
 62. The T cell activating bispecific antigenbinding molecule of claim 1 for use in the treatment of a disease in anindividual in need thereof.
 63. The T cell activating bispecific antigenbinding molecule of claim 62, wherein the disease is cancer.
 64. The Tcell activating bispecific antigen binding molecule of claim 31 for usein the treatment of a disease in an individual in need thereof.
 65. TheT cell activating bispecific antigen binding molecule of claim 33 foruse in the treatment of a disease in an individual in need thereof. 66.The T cell activating bispecific antigen binding molecule of claim 53for use in the treatment of a disease in an individual in need thereof.67. A method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule of claim 1 in a pharmaceutically acceptable form.
 68. Themethod of claim 67, wherein said disease is cancer.
 69. A method oftreating a disease in an individual, comprising administering to saidindividual a therapeutically effective amount of a compositioncomprising the T cell activating bispecific antigen binding molecule ofclaim 31 in a pharmaceutically acceptable form.
 70. A method of treatinga disease in an individual, comprising administering to said individuala therapeutically effective amount of a composition comprising the Tcell activating bispecific antigen binding molecule of claim 33 in apharmaceutically acceptable form.
 71. A method of treating a disease inan individual, comprising administering to said individual atherapeutically effective amount of a composition comprising the T cellactivating bispecific antigen binding molecule of claim 53 in apharmaceutically acceptable form.
 72. A method for inducing lysis of atarget cell, comprising contacting a target cell with the T cellactivating bispecific antigen binding molecule of claim 1 in thepresence of a T cell.
 73. A method for inducing lysis of a target cell,comprising contacting a target cell with the T cell activatingbispecific antigen binding molecule of claim 31 in the presence of a Tcell.
 74. A method for inducing lysis of a target cell, comprisingcontacting a target cell with the T cell activating bispecific antigenbinding molecule of claim 33 in the presence of a T cell.
 75. A methodfor inducing lysis of a target cell, comprising contacting a target cellwith the T cell activating bispecific antigen binding molecule of claim53 in the presence of a T cell.
 76. The T cell activating bispecificantigen binding molecule of claim 16, wherein the third antigen bindingmoiety is an antigen binding moiety capable of specific binding to CEAand comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 23 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:
 27. 77. The T cell activating bispecific antigen bindingmolecule of claim 16, wherein the third antigen binding moiety is anantigen binding moiety capable of specific binding to MCSP and comprisesa heavy chain variable region comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from the group of SEQ ID NO: 13, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 41 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group of SEQ ID NO: 17, SEQ ID NO: 43, SEQ ID NO: 46,SEQ ID NO: 47 and SEQ ID NO:
 51. 78. The T cell activating bispecificantigen binding molecule of claim 21, wherein the Fab light chain of thefirst antigen binding moiety and the Fab light chain of the secondantigen binding moiety are fused to each other, optionally via a peptidelinker.
 79. A host cell comprising the polynucleotide of claim 46.